TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Transition to a mesenchymal state in neuroblastoma may be characterized by a high expression of GD2 and by the acquisition of immune escape from NK cells.

Background: Neuroblastoma (NB) is characterized by both adrenergic (ADRN) and undifferentiated mesenchymal (MES) subsets. The ganglioside sialic acid-containing glycosphingolipid (GD2) is widely overexpressed on tumors of neuroectodermal origin promoting malignant phenotypes. MES cells are greatly enriched in post-therapy and relapsing tumors and are characterized by decreased expression of GD2. This event may cause failure of GD2-based immunotherapy. NK cells represent a key innate cell subset able to efficiently kill tumors. However, the tumor microenvironment (TME) that includes tumor cells and tumor-associated (TA) cells could inhibit their effector function. Methods: We studied eight NB primary cultures that, in comparison with commercial cell lines, more faithfully reflect the tumor cell characteristics. We studied four primary NB-MES cell cultures and two pairs of MES/ADRN (691 and 717) primary cultures, derived from the same patient. In particular, in the six human NB primary cultures, we assessed their phenotype, the expression of GD2, and the enzymes that control its expression, as well as their interactions with NK cells, using flow cytometry, RT-qPCR, and cytotoxicity assays. Results: We identified mature (CD105+/CD133-) and undifferentiated (CD133+/CD105-) NB subsets that express high levels of the MES transcripts WWTR1 and SIX4. In addition, undifferentiated MES cells display a strong resistance to NK-mediated killing. On the contrary, mature NB-MES cells display an intermediate resistance to NK-mediated killing and exhibit some immunomodulatory capacities on NK cells but do not inhibit their cytolytic activity. Notably, independent from their undifferentiated or mature phenotype, NB-MES cells express GD2 that can be further upregulated in undifferentiated NB-MES cells upon co-culture with NK cells, leading to the generation of mature mesenchymal GD2bright neuroblasts. Concerning 691 and 717, they show high levels of GD2 and resistance to NK cell-mediated killing that can be overcome by the administration of dinutuximab beta, the anti-GD2 monoclonal antibody applied in the clinic. Conclusions: NB is a heterogeneous tumor representing a further hurdle in NB immunotherapy. However, different from what was reported with NB commercial cells and independent of their MES/ADRN phenotype, the expression of GD2 and its displayed sensitivity to anti-GD2 mAb ADCC indicated the possible effectiveness of anti-GD2 immunotherapy.

The roles of different forms of IL-15 in human melanoma progression.

Background: Melanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development. Methods: The expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS). Results: Analysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV. Conclusions: Membrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism.

A pathogenic integrated view explaining the different endotypes of asthma and allergic disorders.

The inflammation of allergic diseases is characterized by a complex interaction between type 2 and type 3 immune responses, explaining clinical symptoms and histopathological patterns. Airborne stimuli activate the mucosal epithelium to release a number of molecules impacting the activity of resident immune and environmental cells. Signals from the mucosal barrier, regulatory cells, and the inflamed tissue are crucial conditions able to modify innate and adaptive effector cells providing the selective homing of eosinophils or neutrophils. The high plasticity of resident T- and innate lymphoid cells responding to external signals is the prerequisite to explain the multiplicity of endotypes of allergic diseases. This notion paved the way for the huge use of specific biologic drugs interfering with pathogenic mechanisms of inflammation. Based on the response of the epithelial barrier, the activity of resident regulatory cells, and functions of structural non-lymphoid environmental cells, this review proposes some immunopathogenic scenarios characterizing the principal endotypes which can be associated with a precise phenotype of asthma. Recent literature indicates that similar concepts can also be applied to the inflammation of other non-respiratory allergic disorders. The next challenges will consist in defining specific biomarker(s) of each endotype allowing for a quick diagnosis and the most effective personalized therapy.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

What we know and still ignore on COVID-19 immune pathogenesis and a proposal based on the experience of allergic disorders.

The coronavirus disease 2019 (COVID-19) pandemic started in March 2020 and caused over 5 million confirmed deaths worldwide as far August 2021. We have been recently overwhelmed by a wide literature on how the immune system recognizes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and contributes to COVID-19 pathogenesis. Although originally considered a respiratory viral disease, COVID-19 is now recognized as a far more complex, multi-organ-, immuno-mediated-, and mostly heterogeneous disorder. Though efficient innate and adaptive immunity may control infection, when the patient fails to mount an adequate immune response at the start, or in advanced disease, a high innate-induced inflammation can lead to different clinical outcomes through heterogeneous compensatory mechanisms. The variability of viral load and persistence, the genetic alterations of virus-driven receptors/signaling pathways and the plasticity of innate and adaptive responses may all account for the extreme heterogeneity of pathogenesis and clinical patterns. As recently applied to some inflammatory disorders as asthma, rhinosinusitis with polyposis, and atopic dermatitis, herein we suggest defining different endo-types and the related phenotypes along COVID-19. Patients should be stratified for evolving symptoms and tightly monitored for surrogate biomarkers of innate and adaptive immunity. This would allow to preventively identify each endo-type (and its related phenotype) and to treat patients precisely with agents targeting pathogenic mechanisms.

Neuroblastoma Tumor-Associated Mesenchymal Stromal Cells Regulate the Cytolytic Functions of NK Cells.

Neuroblastoma tumor-associated mesenchymal stromal cells (NB-TA-MSC) have been extensively characterized for their pro-tumorigenic properties, while their immunosuppressive potential, especially against NK cells, has not been thoroughly investigated. Herein, we study the immune-regulatory potential of six primary young and senescent NB-TA-MSC on NK cell function. Young cells display a phenotype (CD105+/CD90+/CD73+/CD29+/CD146+) typical of MSC cells and, in addition, express high levels of immunomodulatory molecules (MHC-I, PDL-1 and PDL-2 and transcriptional-co-activator WWTR1), able to hinder NK cell activity. Notably, four of them express the neuroblastoma marker GD2, the most common target for NB immunotherapy. From a functional point of view, young NB-TA-MSC, contrary to the senescent ones, are resistant to activated NK cell-mediated lysis, but this behavior is overcome using anti-CD105 antibody TRC105 that activates antibody-dependent cell-mediated cytotoxicity. In addition, proliferating NB-TA-MSC, but not the senescent ones, after six days of co-culture, inhibit proliferation, expression of activating receptors and cytolytic activity of freshly isolated NK. Inhibitors of the soluble immunosuppressive factors L-kynurenine and prostaglandin E2 efficiently counteract this latter effect. Our data highlight the presence of phenotypically heterogeneous NB-TA-MSC displaying potent immunoregulatory properties towards NK cells, whose inhibition could be mandatory to improve the antitumor efficacy of targeted immunotherapy.

Pathogenic Mechanisms of Vaccine-Induced Immune Thrombotic Thrombocytopenia in People Receiving Anti-COVID-19 Adenoviral-Based Vaccines: A Proposal.

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.

Pediatric Tumors-Mediated Inhibitory Effect on NK Cells: The Case of Neuroblastoma and Wilms' Tumors.

Natural killer (NK) cells play a key role in the control of cancer development, progression and metastatic dissemination. However, tumor cells develop an array of strategies capable of impairing the activation and function of the immune system, including NK cells. In this context, a major event is represented by the establishment of an immunosuppressive tumor microenvironment (TME) composed of stromal cells, myeloid-derived suppressor cells, tumor-associated macrophages, regulatory T cells and cancer cells themselves. The different immunoregulatory cells infiltrating the TME, through the release of several immunosuppressive molecules or by cell-to-cell interactions, cause an impairment of the recruitment of NK cells and other lymphocytes with effector functions. The different mechanisms by which stromal and tumor cells impair NK cell function have been particularly explored in adult solid tumors and, in less depth, investigated and discussed in a pediatric setting. In this review, we will compare pediatric and adult solid malignancies concerning the respective mechanisms of NK cell inhibition, highlighting novel key data in neuroblastoma and Wilms' tumor, two of the most frequent pediatric extracranial solid tumors. Indeed, both tumors are characterized by the presence of stromal cells acting through the release of immunosuppressive molecules. In addition, specific tumor cell subsets inhibit NK cell cytotoxic function by cell-to-cell contact mechanisms likely controlled by the transcriptional coactivator TAZ. These findings could lead to a more performant diagnostic approach and to the development of novel immunotherapeutic strategies targeting the identified cellular and molecular targets.

Stromal-like Wilms tumor cells induce human Natural Killer cell degranulation and display immunomodulatory properties towards NK cells.

The similarity of stromal-like Wilms tumor (str-WT) cells with mesenchymal stem cells (MSC), suggests their relevant role in the interplay with immune cells in the tumor microenvironment. We investigated the interaction between str-WT cells and NK cells. We observed that str-WT cells expressed some major ligands for activating and inhibitory NK cell receptors. Moreover, they expressed inhibitory checkpoint molecules involved in the negative regulation of anti-tumor immune response. The analysis of the interaction between str-WT cells and NK lymphocytes revealed that activated NK cells could efficiently degranulate upon interaction with str-WT cells. On the other hand, str-WT cells could exert potent inhibitory effects on cytokine-induced activation of NK cell proliferation and phenotype, which were mediated by the production of IDO and PGE2 inhibitory factors. Our data provide insight into the molecular interactions between str-WT cells and NK lymphocytes that may result in different outcomes possibly occurring in the WT microenvironment.

Identification of neuroblastoma cell lines with uncommon TAZ+/mesenchymal stromal cell phenotype with strong suppressive activity on natural killer cells.

BACKGROUND: Neuroblastoma (NB) is the most common, extracranial childhood solid tumor arising from neural crest progenitor cells and is a primary cause of death in pediatric patients. In solid tumors, stromal elements recruited or generated by the cancer cells favor the development of an immune-suppressive microenvironment. Herein, we investigated in NB cell lines and in NB biopsies, the presence of cancer cells with mesenchymal phenotype and determined the immune-suppressive properties of these tumor cells on natural killer (NK) cells. METHODS: We assessed the mesenchymal stromal cell (MSC)-like phenotype and function of five human NB cell lines and the presence of this particular subset of neuroblasts in NB biopsies using flow-cytometry, immunohistochemistry, RT-qPCR, cytotoxicity assays, western blot and silencing strategy. We corroborated our data consulting a public gene-expression dataset. RESULTS: Two NB cell lines, SK-N-AS and SK-N-BE(2)C, exhibited an unprecedented MSC phenotype (CD105+/CD90+/CD73+/CD29+/CD146+/GD2+/TAZ+). In these NB-MSCs, the ectoenzyme CD73 and the oncogenic/immune-regulatory transcriptional coactivator TAZ were peculiar markers. Their MSC-like nature was confirmed by their adipogenic and osteogenic differentiation potential. Immunohistochemical analysis confirmed the presence of neuroblasts with MSC phenotype (CD105+/CD73+/TAZ+). Moreover, a public gene-expression dataset revealed that, in stage IV NB, a higher expression of TAZ and CD105 strongly correlated with a poorer outcome.Among the NB-cell lines analyzed, only NB-MSCs exhibited multifactorial resistance to NK-mediated lysis, inhibition of activating NK receptors, signal adaptors and of NK-cell cytotoxicity through cell-cell contact mediated mechanisms. The latter property was controlled partially by TAZ, since its silencing in NB cells efficiently rescued NK-cell cytotoxic activity, while its overexpression induced opposite effects in non-NB-MSC cells. CONCLUSIONS: We identified a novel NB immunoregulatory subset that: (i) displayed phenotypic and functional properties of MSC, (ii) mediated multifactorial resistance to NK-cell-induced killing and (iii) efficiently inhibited, in coculture, the cytotoxic activity of NK cells against target cells through a TAZ-dependent mechanism. These findings indicate that targeting novel cellular and molecular components may disrupt the immunomodulatory milieu of the NB microenvironment ameliorating the response to conventional treatments as well as to advanced immunotherapeutic approaches, including adoptive transfer of NK cells and chimeric antigen receptor T or NK cells.

Wilms' Tumor Primary Cells Display Potent Immunoregulatory Properties on NK Cells and Macrophages.

The immune response plays a crucial defensive role in cancer growth and metastasis and is a promising target in different tumors. The role of the immune system in Wilm's Tumor (WT), a common pediatric renal malignancy, is still to be explored. The characterization of the immune environment in WT could allow the identification of new therapeutic strategies for targeting possible inhibitory mechanisms and/or lowering toxicity of the current treatments. In this study, we stabilized four WT primary cultures expressing either a blastematous (CD56+/CD133-) or an epithelial (CD56-/CD133+) phenotype and investigated their interactions with innate immune cells, namely NK cells and monocytes. We show that cytokine-activated NK cells efficiently kill WT cells. However, after co-culture with WT primary cells, NK cells displayed an impaired cytotoxic activity, decreased production of IFNγ and expression of CD107a, DNAM-1 and NKp30. Analysis of the effects of the interaction between WT cells and monocytes revealed their polarization towards alternatively activated macrophages (M2) that, in turn, further impaired NK cell functions. In conclusion, we show that both WT blastematous and epithelial components may contribute directly and indirectly to a tumor immunosuppressive microenvironment that is likely to play a role in tumor progression.

Interleukin-15 and cancer: some solved and many unsolved questions.

Soluble interleukin (IL)-15 exists under two forms: as monomer (sIL-15) or as heterodimeric complex in association with sIL-15Rα (sIL-15/IL-15Rα). Both forms have been successfully tested in experimental tumor murine models and are currently undergoing investigation in phase I/II clinical trials. Despite more than 20 years research on IL-15, some controversial issues remain to be addressed. A first point concerns the detection of the sIL-15/IL-15Rα in plasma of healthy donors or patients with cancer and its biological significance. The second and third unsolved question regards the protumorigenic role of the IL-15/IL-15Rα complex in human cancer and the detrimental immunological consequences associated to prolonged exposure of natural killer (NK) cells to both forms of soluble IL-15, respectively. Data suggest that in vivo prolonged or repeated exposure to monomeric sIL-15 or the soluble complex may lead to NK hypo-responsiveness through the expansion of the CD8+/CD44+ T cell subset that would suppress NK cell functions. In vitro experiments indicate that soluble complex and monomeric IL-15 may cause NK hyporesponsiveness through a direct effect caused by their prolonged stimulation, suggesting that this mechanism could also be effective in vivo. Therefore, a better knowledge of IL-15 and a more appropriate use of both its soluble forms, in terms of concentrations and time of exposure, are essential in order to improve their therapeutic use. In cancer, the overproduction of sIL-15/IL-15Rα could represent a novel mechanism of immune escape. The soluble complex may act as a decoy cytokine unable to efficiently foster NK cells, or could induce NK hyporesponsiveness through an excessive and prolonged stimulation depending on the type of IL-15Rα isoforms associated. All these unsolved questions are not merely limited to the knowledge of IL-15 pathophysiology, but are crucial also for the therapeutic use of this cytokine. Therefore, in this review, we will discuss key unanswered issues on the heterogeneity and biological significance of IL-15 isoforms, analyzing both their cancer-related biological functions and their therapeutic implications.

Inhibition of TGF-ß1 Signaling by IL-15: A Novel Role for IL-15 in the Control of Renal Epithelial-Mesenchymal Transition: IL-15 Counteracts TGF-ß1-Induced EMT in Renal Fibrosis.

Renal tubulointerstitial fibrosis is the final common pathway in end-stage renal disease and is characterized by aberrant accumulation of extracellular matrix (ECM) components secreted by myofibroblasts. Tubular type 2 EMT, induced by TGF-ß, plays an important role in renal fibrosis, by participating directly or indirectly in myofibroblasts generation. TGF-ß1-induced apoptosis and fibrosis in experimental chronic murine kidney diseases are concomitantly associated with an intrarenal decreased expression of the IL-15 survival factor. Since IL-15 counteracts TGF-ß1 effects in different cell models, we analyzed whether (1) human chronic inflammatory nephropathies evolving towards fibrosis could be also characterized by a weak intrarenal IL-15 expression and (2) IL-15 could inhibit epithelial-mesenchymal transition (EMT) and excess matrix deposition in human renal proximal tubular epithelial cells (RPTEC). Our data show that different human chronic kidney diseases are characterized by a strong decreased expression of intrarenal IL-15, which is particularly relevant in diabetic nephropathy, in which type 2 tubular EMT plays an important role in fibrosis. Moreover, primary epithelial tubular cultures deprived of growth supplements rapidly produce active TGF-ß1 inducing a "spontaneous" EMT process characterized by the loss of membrane-bound IL-15 (mbIL-15) expression. Both "spontaneous" EMT and recombinant human (rh) TGF-ß1-induced EMT models can be inhibited by treating RPTEC and HK2 cells with rhIL-15. Through a long-lasting phospho-c-jun activation, IL-15 inhibits rhTGF-ß1-induced Snail1 expression, the master inducer of EMT, and blocks TGF-ß1-induced tubular EMT and downstream collagen synthesis. In conclusion, our data suggest that intrarenal IL-15 could be a natural inhibitor of TGF-ß in human kidney able to guarantee epithelial homeostasis and to prevent EMT process. Thus, both in vivo and in vitro an unbalance in intrarenal IL-15 and TGF-ß1 levels could render RPTEC cells more prone to undergo EMT process. Exogenous IL-15 treatment could be beneficial in some human nephropathies such as diabetic nephropathy.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer.

BACKGROUND: Cancer cells from different origins exhibit various basal redox statuses and thus respond differently to intrinsic or extrinsic oxidative stress. These intricate characteristics condition the success of redox-based anticancer therapies that capitalize on the ability of reactive oxygen species to achieve selective and efficient cancer cell killing. METHODS: Redox biology methods, stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, and bioinformatics pattern comparisons were used to decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and vitamin C (VC). The in vivo effect of AUF, VC, and two AUF/VC combinations on mice bearing MDA-MB-231 xenografts (n = 5 mice per group) was also evaluated. All statistical tests were two-sided. RESULTS: AUF targeted simultaneously the thioredoxin and glutathione antioxidant systems. AUF/VC combinations exerted a synergistic and hydrogen peroxide (H2O2)-mediated cytotoxicity toward MDA-MB-231 cells and other breast cancer cell lines. The anticancer potential of AUF/VC combinations was validated in vivo on MDA-MB-231 xenografts in mice without notable side effects. On day 14 of treatments, mean (SD) tumor volumes for the vehicle-treated control group and the two AUF/VC combination-treated groups (A/V1 and A/V2) were 197.67 (24.28) mm3, 15.66 (10.90) mm3, and 10.23 (7.30)mm3, respectively; adjusted P values of the differences between mean tumor volumes of vehicle vs A/V1 groups and vehicle vs A/V2 groups were both less than .001. SILAC proteomics, bioinformatics analysis, and functional experiments linked prostaglandin reductase 1 (PTGR1) expression levels with breast cancer cell sensitivity to AUF/VC combinations. CONCLUSION: The combination of AUF and VC, two commonly available drugs, could be efficient against triple-negative breast cancer and potentially other cancers with similar redox properties and PTGR1 expression levels. The redox-based anticancer activity of this combination and the discriminatory potential of PTGR1 expression are worth further assessment in preclinical and clinical studies.

Isolation and characterization of renal cancer stem cells from patient-derived xenografts.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133- over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.

Human Renal Normal, Tumoral, and Cancer Stem Cells Express Membrane-Bound Interleukin-15 Isoforms Displaying Different Functions.

Intrarenal interleukin-15 (IL-15) participates to renal pathophysiology, but the role of its different membrane-bound isoforms remains to be elucidated. In this study, we reassess the biology of membrane-bound IL-15 (mb-IL-15) isoforms by comparing primary cultures of human renal proximal tubular epithelial cells (RPTEC) to peritumoral (ptumTEC), tumoral (RCC), and cancer stem cells (CSC/CD105(+)). RPTEC express a 14 to 16 kDa mb-IL-15, whose existence has been assumed but never formally demonstrated and likely represents the isoform anchored at the cell membrane through the IL-15 receptor α (IL-15Rα) chain, because it is sensitive to acidic treatment and is not competent to deliver a reverse signal. By contrast, ptumTEC, RCC, and CSC express a novel N-hyperglycosylated, short-lived transmembrane mb-IL-15 (tmb-IL-15) isoform around 27 kDa, resistant to acidic shock, delivering a reverse signal in response to its soluble receptor (sIL-15Rα). This reverse signal triggers the down-regulation of the tumor suppressor gene E-cadherin in ptumTEC and RCC but not in CSC/CD105(+), where it promotes survival. Indeed, through the AKT pathway, tmb-IL-15 protects CSC/CD105(+) from non-programmed cell death induced by serum starvation. Finally, both mb-IL-15 and tmb-IL-15 are sensitive to metalloproteases, and the cleaved tmb-IL-15 (25 kDa) displays a powerful anti-apoptotic effect on human hematopoietic cells. Overall, our data indicate that both mb-IL-15 and tmb-IL-15 isoforms play a complex role in renal pathophysiology downregulating E-cadherin and favoring cell survival. Moreover, "apparently normal" ptumTEC cells, sharing different properties with RCC, could contribute to organize an enlarged peritumoral "preneoplastic" environment committed to favor tumor progression.

Cannabinoid receptor 1 is a major mediator of renal fibrosis.

Chronic kidney disease, secondary to renal fibrogenesis, is a burden on public health. There is a need to explore new therapeutic pathways to reduce renal fibrogenesis. To study this, we used unilateral ureteral obstruction (UUO) in mice as an experimental model of renal fibrosis and microarray analysis to compare gene expression in fibrotic and normal kidneys. The cannabinoid receptor 1 (CB1) was among the most upregulated genes in mice, and the main endogenous CB1 ligand (2-arachidonoylglycerol) was significantly increased in the fibrotic kidney. Interestingly, CB1 expression was highly increased in kidney biopsies of patients with IgA nephropathy, diabetes, and acute interstitial nephritis. Both genetic and pharmacological knockout of CB1 induced a profound reduction in renal fibrosis during UUO. While CB2 is also involved in renal fibrogenesis, it did not potentiate the role of CB1. CB1 expression was significantly increased in myofibroblasts, the main effector cells in renal fibrogenesis, upon TGF-ß1 stimulation. The decrease in renal fibrosis during CB1 blockade could be explained by a direct action on myofibroblasts. CB1 blockade reduced collagen expression in vitro. Rimonabant, a selective CB1 endocannabinoid receptor antagonist, modulated the macrophage infiltrate responsible for renal fibrosis in UUO through a decrease in monocyte chemoattractant protein-1 synthesis. Thus, CB1 has a major role in the activation of myofibroblasts and may be a new target for treating chronic kidney disease.

TLR ligands stimulation protects MSC from NK killing.

Mesenchymal stem cells (MSCs) play a fundamental role in allograft rejection and graft-versus-host disease through their immunosuppressive abilities. Recently, Toll-like receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and natural killer (NK) cells. Our results show that TLR-primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL-2-activated NK-induced killing. Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility complex class I chain A and ULBP3 and DNAM-1 ligands by TLR-primed MSC. These results indicate that MSCs are able to adapt their immuno-behavior in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4-primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC-mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders.