T Cells in Colorectal Cancer: Unravelling the Function of Different T Cell Subsets in the Tumor Microenvironment.

Therapeutic options for metastatic colorectal cancer (mCRC) are very limited, and the prognosis using combination therapy with a chemotherapeutic drug and a targeted agent, e.g., epidermal growth factor receptor or tyrosine kinase, remains poor. Therefore, mCRC is associated with a poor median overall survival (mOS) of only 25-30 months. Current immunotherapies with checkpoint inhibitor blockade (ICB) have led to a substantial change in the treatment of several cancers, such as melanoma and non-small cell lung cancer. In CRC, ICB has only limited effects, except in patients with microsatellite instability-high (MSI-H) or mismatch repair-deficient (dMMR) tumors, which comprise about 15% of sporadic CRC patients and about 4% of patients with metastatic CRC. The vast majority of sporadic CRCs are microsatellite-stable (MSS) tumors with low levels of infiltrating immune cells, in which immunotherapy has no clinical benefit so far. Immunotherapy with checkpoint inhibitors requires the presence of infiltrating T cells into the tumor microenvironment (TME). This makes T cells the most important effector cells in the TME, as evidenced by the establishment of the immunoscore-a method to estimate the prognosis of CRC patients. The microenvironment of a tumor contains several types of T cells that are anti-tumorigenic, such as CD8+ T cells or pro-tumorigenic, such as regulatory T cells (Tregs) or T helper 17 (Th17) cells. However, even CD8+ T cells show marked heterogeneity, e.g., they can become exhausted, enter a state of hyporesponsiveness or become dysfunctional and express high levels of checkpoint molecules, the targets for ICB. To kill cancer cells, CD8+ T cells need the recognition of the MHC class I, which is often downregulated on colorectal cancer cells. In this case, a population of unconventional T cells with a γδ T cell receptor can overcome the limitations of the conventional CD8+ T cells with an αßT cell receptor. γδ T cells recognize antigens in an MHC-independent manner, thus acting as a bridge between innate and adaptive immunity. Here, we discuss the effects of different T cell subsets in colorectal cancer with a special emphasis on γδ T cells and the possibility of using them in CAR-T cell therapy. We explain T cell exclusion in microsatellite-stable colorectal cancer and the possibilities to overcome this exclusion to enable immunotherapy even in these "cold" tumors.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

T-helper-1-cell cytokines drive cancer into senescence.

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Immune checkpoint blockade therapy.

Immune checkpoints are accessory molecules that either promote or inhibit T-cell activation. Two inhibitory molecules, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), got high attention, as inhibition of CTLA-4 or PD-1 signaling provides the first immune therapy that significantly improves the survival of patients with metastatic solid cancers. Inhibition of CTLA-4 or PD-1 was first studied in and approved for patients with metastatic melanoma. Blocking immune checkpoints is also efficient in non-small-cell lung cancer, renal cell cancers, hypermutated gastrointestinal cancers, and others. Immune responses, whether directed against infections or against tumors, are divided into 2 phases: an initiation phase and an activation phase, where the immune system recognizes a danger signal and becomes activated by innate signals to fight the danger. This reaction is fundamental for the control of infections and cancer, but needs to be turned off once the danger is controlled, because persistence of this activation ultimately causes severe tissue damage. Therefore, each activation of the immune system is followed by a termination phase, where endogenous immune suppressor molecules arrest immune responses to prevent harmful damage. In the case of cancer immune therapies, therapeutic approaches classically enhanced the initiation and activation of immune responses to increase the emergence and the efficacy of cytotoxic T lymphocytes (CTL) against cancers. In sharp contrast, immune checkpoint blockade focuses on the termination of immune responses by inhibiting immune suppressor molecules. It thus prevents the termination of immune responses or even awakes those CTLs that became exhausted during an immune response. Therefore, blocking negatively regulating immune checkpoints restores the capacity of exhausted CTL to kill the cancer they infiltrate. In addition, they drive surviving cancer cells into a still poorly defined state of dormancy. As the therapy also awakes self-reactive CTL, one downside of the therapy is the induction of organ-specific autoimmune diseases. The second downside is the exorbitant drug price that withdraws patients in need from a therapy that was developed by academic research, which impairs further academic treatment development and financially charges the public health system.

Cytokine-induced senescence for cancer surveillance.

The immune response is a first-line systemic defense to curb tumorigenesis and metastasis. Much effort has been invested to design antitumor interventions that would boost the immune system in its fight to defeat or contain cancerous growth. Tumor vaccination protocols, transfer of tumor-associated-antigen-specific T cells, T cell activity-regulating antibodies, and recombinant cytokines are counted among a toolbox filled with immunotherapeutic options. Although the mechanistic underpinnings of tumor immune control remain to be deciphered, these are studied with the goal of cancer cell destruction. In contrast, tumor dormancy is considered as a dangerous equilibrium between cell proliferation and cell death. There is, however, emerging evidence that tumor immune control can be achieved in the absence of overt cancer cell death. Here, we propose cytokine-induced senescence (CIS) by transfer of T helper-1 cells (TH1) or by recombinant cytokines as a novel therapeutic intervention for cancer treatment. Immunity-induced senescence triggers a stable cell cycle arrest of cancer cells. It engages the immune system to construct defensive, isolating barriers around tumors, and prevents tumor growth through the delivery or induction of TH1-cytokines in the tumor microenvironment. Keeping cancer cells in a non-proliferating state is a strategy, which directly copes with the lost homeostasis of aggressive tumors. As most studies show that even after efficient cancer therapies minimal residual disease persists, we suggest that therapies should include immune-mediated senescence for cancer surveillance. CIS has the goal to control the residual tumor and to transform a deadly disease into a state of silent tumor persistence.

Nuclear Translocation of Argonaute 2 in Cytokine-Induced Senescence.

BACKGROUND/AIMS: Cellular senescence, or permanent growth arrest, is known as an effective tumor suppressor mechanism that can be induced by different stressors, such as oncogenes, chemotherapeutics or cytokine cocktails. Previous studies demonstrated that the growth-repressing state of oncogene-induced senescent cells depends on argonaute protein 2 (Ago2)-mediated transcriptional gene silencing and Ago2/Rb corepression of E2F-dependent cell cycle genes. Cytokine-induced senescence (CIS) likewise depends on activation of the p16Ink4a/Rb pathway, and consecutive inactivation of the E2F family of transcription factors. In the present study, we therefore analyzed the role of Ago2 in CIS. METHODS: Human cancer cell lines were treated with interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) to induce senescence. Senescence was determined by growth assays and measurement of senescence-associated ß-galactosidase (SA-ß-gal) activity, Ago2 translocation by Ago2/ Ki67 immunofluorescence staining and western blot analysis, and gene transcription by quantitative polymerase chain reaction (qPCR). RESULTS: IFN-γ and TNF permanently stopped cell proliferation and time-dependently increased SA-ß-gal activity. After 24 - 48 h of cytokine treatment, Ago2 translocated from the cytoplasm into the nucleus of Ki67-negative cells, an effect which was shown to be reversible. Importantly, the proinflammatory cytokine cocktail suppressed Ago2-regulated cell cycle control genes, and siRNA-mediated depletion of Ago2 interfered with cytokine-induced growth inhibition. CONCLUSION: IFN-γ and TNF induce a stable cell cycle arrest of cancer cells that is accompanied by a fast nuclear Ago2 translocation and repression of Ago2-regulated cell cycle control genes. As Ago2 downregulation impairs cytokine-induced growth regulation, Ago2 may contribute to tissue homeostasis in human cancers.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Immunotherapy of melanoma: efficacy and mode of action.

Forty years of research have brought about the development of antibodies that induce effective antitumor immune responses through sustained activation of the immune system. These "immune checkpoint inhibitors" are directed against immune inhibitory molecules, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1) or programmed death ligand 1 (PD-L1). Disruption of the PD-1/PD-L1 interaction improves the intermediate-term prognosis even in patients with advanced stage IV melanoma. One and a half years after treatment initiation, 30-60 % of these patients are still alive. While cancer immunotherapies usually do not eradicate metastases completely, they do cause a regression by 20-80 %. It is well established that the immune system is able to kill tumor cells, and this has also been demonstrated for immunotherapies. Preclinical data, however, has shown that anti-cancer immunity is not limited to killing cancer cells. Thus, through interferon gamma and tumor necrosis factor, the immune system is able to induce stable tumor growth arrest, referred to as senescence. Ensuring patient survival by long-term stabilization of metastatic growth will therefore become a central goal of antitumor immunotherapies. This therapeutic approach is effective in melanoma and non-small-cell lung cancer. Once immunotherapies also have an indication for common cancer types, drug prices will have to drop considerably in order to be able to keep them available to those dependent on such therapies.

Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide.

Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

Effective T-cell recall responses require the taurine transporter Taut.

T-cell activation and the subsequent transformation of activated T cells into T-cell blasts require profound changes in cell volume. However, the impact of cell volume regulation for T-cell immunology has not been characterized. Here we studied the role of the cell-volume regulating osmolyte transporter Taut for T-cell activation in Taut-deficient mice. T-cell mediated recall responses were severely impaired in taut(-/-) mice as shown with B16 melanoma rejection and hapten-induced contact hypersensitivity. CD4(+) and CD8(+) T cells were unequivocally located within peripheral lymph nodes of unprimed taut(-/-) mice but significantly decreased in taut(-/-) compared with taut(+/+) mice following in vivo activation. Further analysis revealed that Taut is critical for rescuing T cells from activation-induced cell death in vitro and in vivo as shown with TCR, superantigen, and antigen-specific activation. Consequently, reduction of CD4(+) and CD8(+) T cells in taut(-/-) mice upon antigen challenge resulted in impaired in vivo generation of T-cell memory. These findings disclose for the first time that volume regulation in T cells is an element in the regulation of adaptive immune responses and that the osmolyte transporter Taut is crucial for T-cell survival and T-cell mediated immune reactions.

Age sensitivity of NFκB abundance and programmed cell death in erythrocytes induced by NFκB inhibitors.

BACKGROUND/AIMS: Erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte outer membrane. Susceptibility to eryptosis is enhanced in aged erythrocytes and stimulated by NFκB-inhibitors Bay 11-7082 and parthenolide. Here we explored whether expression of NFκB and susceptibility to inhibitor-induced eryptosis is sensitive to erythrocyte age. METHODS: Human erythrocytes were separated into five fractions, based on age-associated characteristics cell density and volume. NFκB compared to ß-actin protein abundance was estimated by Western blotting and cell volume from forward scatter. Phosphatidylserine exposure was identified using annexin-V binding. RESULTS: NFκB was most abundant in young erythrocytes but virtually absent in aged erythrocytes. A 24h or 48h exposure to Ringer resulted in spontaneous decrease of forward scatter and increase of annexin V binding, effects more pronounced in aged than in young erythrocytes. Both, Bay 11-7082 (20 µM) and parthenolide (100 µM) triggered eryptosis, effects again most pronounced in aged erythrocytes. CONCLUSION: NFκB protein abundance is lowest and spontaneous eryptosis as well as susceptibility to Bay 11-7082 and parthenolide highest in aged erythrocytes. Thus, inhibition of NFκB signalling alone is not responsible for the stimulation of eryptosis by parthenolide or Bay 11-7082.

Nucleoside analogues with a 1,3-diene-Fe(CO)3 substructure: stereoselective synthesis, configurational assignment, and apoptosis-inducing activity.

The synthesis and stereochemical assignment of two classes of iron-containing nucleoside analogues, both of which contain a butadiene-Fe(CO)3 substructure, is described. The first type of compounds are Fe(CO)3-complexed 3'-alkenyl-2',3'-dideoxy-2',3'-dehydro nucleosides (2,5-dihydrofuran derivatives), from which the second class of compounds is derived by formal replacement of the ring oxygen atom by a CH2 group (carbocyclic nucleoside analogues). These compounds were prepared in a stereoselective manner through the metal-assisted introduction of the nucleobase. Whilst the furanoid intermediates were prepared from carbohydrates (such as methyl-glucopyranoside), the carbocyclic compounds were obtained by using an intramolecular Pauson-Khand reaction. Stereochemical assignments based on NMR and CD spectroscopy were confirmed by X-ray structural analysis. Biological investigations revealed that several of the complexes exhibited pronounced apoptosis-inducing properties (through an unusual caspase 3-independent but ROS-dependent pathway). Furthermore, some structure-activity relationships were identified, also as a precondition for the design and synthesis of fluorescent and biotin-labeled conjugates.

Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion.

Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET.Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms.Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography.Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC.Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.

The impact of erythrocyte age on eryptosis.

Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age-associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca(2+) level from Fluo-3-dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N-acetyl-L-cysteine in vitro. Also, N-acetyl-L-cysteine significantly prolonged the half-life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.

Potential roles of the NFκB and glutathione pathways in mature human erythrocytes.

Anucleated erythrocytes were long considered as oxygen-transporting cells with limited regulatory functions. Components of different nuclear signaling pathways have not been investigated in those cells, yet. Surprisingly, we repeatedly found significant amounts of transcription factors in purified erythrocyte preparations, i.e. nuclear factor κB (NFκB), and major components of the canonical NFκB signaling pathway. To investigate the functional role of NFκB signaling, the effects of the preclinical compounds Bay 11-7082 and parthenolide on the survival of highly purified erythrocytes were investigated. Interestingly, both inhibitors of the NFκB pathway triggered erythrocyte programmed cell death as demonstrated by enhanced phospholipid scrambling (phosphatidylserine exposure) and cell shrinkage. Anucleated erythrocytes are an ideal cellular model allowing the study of nongenomic mechanisms contributing to suicidal cell death. As NFκB inhibitors might also interfere with the anti-oxidative defense systems of the cell, we measured the levels of reduced glutathione (GSH) after challenge with the inhibitors. Indeed, incubation of erythrocytes with Bay 11-7082 clearly decreased erythrocyte GSH levels. In conclusion, the pharmacological inhibitors of the NFκB pathway Bay 11-7082 and parthenolide interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression. Besides affecting erythrocyte survival, NFκB inhibition and induction of erythrocyte phosphatidylserine exposure may influence blood clotting. Future studies will be aimed at discriminating between NFκB-dependent and NFκB-independent GSH-mediated effects of Bay 11-7082 and parthenolide on erythrocyte death.

Eryptosis: Programmed Death of Nucleus-Free, Iron-Filled Blood Cells.

Human erythrocytes are organelle-free cells packaged with iron-containing hemoglobin, specializing in the transport of oxygen. With a total number of approximately 25 trillion cells per individual, the erythrocyte is the most abundant cell type not only in blood but in the whole organism. Despite their low complexity and their inability to transcriptionally upregulate antioxidant defense mechanisms, they display a relatively long life time, of 120 days. This ensures the maintenance of tissue homeostasis where the clearance of old or damaged erythrocytes is kept in balance with erythropoiesis. Whereas the regulatory mechanisms of erythropoiesis have been elucidated over decades of intensive research, the understanding of the mechanisms of erythrocyte clearance still requires some refinement. Here, we present the main pathways leading to eryptosis, the programmed death of erythrocytes, with special emphasis on Ca2+ influx, the generation of ceramide, oxidative stress, kinase activation, and iron metabolism. We also compare stress-induced erythrocyte death with erythrocyte ageing and clearance, and discuss the similarities between eryptosis and ferroptosis, the iron-dependent regulated death of nucleated blood cells. Finally, we focus on the pathologic consequences of deranged eryptosis, and discuss eryptosis in the context of different infectious diseases, e.g., viral or parasitic infections, and hematologic disorders.

Cytokine-Induced Senescence in the Tumor Microenvironment and Its Effects on Anti-Tumor Immune Responses.

In contrast to surgical excision, chemotherapy or radiation therapy, immune checkpoint blockade therapies primarily influence cells in the tumor microenvironment, especially the tumor-associated lymphocytes and antigen-presenting cells. Besides complete remission of tumor lesions, in some patients, early tumor regression is followed by a consolidation phase where residing tumors remain dormant. Whereas the cytotoxic mechanisms of the regression phase (i.e., apoptosis, necrosis, necroptosis, and immune cell-mediated cell death) have been extensively described, the mechanisms underlying the dormant state are still a matter of debate. Here, we propose immune-mediated induction of senescence in cancers as one important player. Senescence can be achieved by tumor-associated antigen-specific T helper 1 cells, cytokines or antibodies targeting immune checkpoints. This concept differs from cytotoxic treatment, which often targets the genetic makeup of cancer cells. The immune system's ability to establish "defensive walls" around tumors also places the tumor microenvironment into the fight against cancer. Those "defensive walls" isolate the tumor cells instead of increasing the selective pressure. They also keep the tumor cells in a non-proliferating state, thereby correcting the derailed tissue homeostasis. In conclusion, strengthening the senescence surveillance of tumors by the immune cells of the microenvironment is a future goal to dampen this life-threatening disease.

IFN-γ and TNF Induce Senescence and a Distinct Senescence-Associated Secretory Phenotype in Melanoma.

Immune checkpoint blockade (ICB) therapy is a central pillar of melanoma treatment leading to durable response rates. Important mechanisms of action of ICB therapy include disinhibition of CD4+ and CD8+ T cells. Stimulated CD4+ T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated ß-galactosidase (SA-ß-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-ß-gal activity, and-except palbociclib-increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.

The Cytokine Network in Colorectal Cancer: Implications for New Treatment Strategies.

Colorectal cancer (CRC) is one of the most frequent tumor entities worldwide with only limited therapeutic options. CRC is not only a genetic disease with several mutations in specific oncogenes and/or tumor suppressor genes such as APC, KRAS, PIC3CA, BRAF, SMAD4 or TP53 but also a multifactorial disease including environmental factors. Cancer cells communicate with their environment mostly via soluble factors such as cytokines, chemokines or growth factors to generate a favorable tumor microenvironment (TME). The TME, a heterogeneous population of differentiated and progenitor cells, plays a critical role in regulating tumor development, growth, invasion, metastasis and therapy resistance. In this context, cytokines from cancer cells and cells of the TME influence each other, eliciting an inflammatory milieu that can either enhance or suppress tumor growth and metastasis. Additionally, several lines of evidence exist that the composition of the microbiota regulates inflammatory processes, controlled by cytokine secretion, that play a role in carcinogenesis and tumor progression. In this review, we discuss the cytokine networks between cancer cells and the TME and microbiome in colorectal cancer and the related treatment strategies, with the goal to discuss cytokine-mediated strategies that could overcome the common therapeutic resistance of CRC tumors.

The NFĸB pathway inhibitors Bay 11-7082 and parthenolide induce programmed cell death in anucleated Erythrocytes.

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFκB, by inhibiting NFκB directly (parthenolide) or by interfering with the inactivation of the NFκB inhibitory protein IκB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca(2+) leakage or glutathione depletion. The present study utilized Western blotting to search for NFκB and IκB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca(2+) (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFκB and IκB-α are expressed in erythrocytes. Targeting the NFκB pathway by Bay 11-7082 (IC(50) ≈ 10 µM) and parthenolide (IC(50) ≈ 30 µM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca(2+) and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFκB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression.