RESUMO
Small cell lung cancer (SCLC) is treated as a homogeneous disease, although the expression of NEUROD1, ASCL1, POU2F3, and YAP1 identifies distinct molecular subtypes. The MYC oncogene, amplified in SCLC, was recently shown to act as a lineage-specific factor to associate subtypes with histological classes. Indeed, MYC-driven SCLCs show a distinct metabolic profile and drug sensitivity. To disentangle their molecular features, we focused on the co-amplified PVT1, frequently overexpressed and originating circular (circRNA) and chimeric RNAs. We analyzed hsa_circ_0001821 (circPVT1) and PVT1/AKT3 (chimPVT1) as examples of such transcripts, respectively, to unveil their tumorigenic contribution to SCLC. In detail, circPVT1 activated a pro-proliferative and anti-apoptotic program when over-expressed in lung cells, and knockdown of chimPVT1 induced a decrease in cell growth and an increase of apoptosis in SCLC in vitro. Moreover, the investigated PVT1 transcripts underlined a functional connection between MYC and YAP1/POU2F3, suggesting that they contribute to the transcriptional landscape associated with MYC amplification. In conclusion, we have uncovered a functional role of circular and chimeric PVT1 transcripts in SCLC; these entities may prove useful as novel biomarkers in MYC-amplified tumors.
Assuntos
Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Neoplasias Pulmonares/genética , Proliferação de Células/genética , Apoptose/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Multiple myeloma (MM) progression and drug resistance depend on the crosstalk between MM cells and bone marrow (BM) fibroblasts (FBs). During monoclonal gammopathy of undetermined significance (MGUS) to MM transition, MM cell-derived exosomes (EXOs) reprogram the miRNA (miR) profile of FBs, inducing the overexpression miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Here, we demonstrate that the miR content of MM FB-derived EXOs (FB-EXOs) overlaps the miR profile of parental FBs by overexpressing comparable levels of miR-23b-3p, miR-27b-3p, miR-125b-5p, miR-214-3p, and miR-5100. Recipient MM cells co-cultured with MM FB-EXOs selectively overexpress only miR-214-3p and miR-5100 but not miR-23b-3p, miR-27b-3p, and miR-125b-5p, suggesting a putative selective transfer. MM cells express HOTAIR, TOB1-AS1, and MALAT1 lncRNAs. Transient transfection of MM cells with lnc·siRNAs demonstrates that HOTAIR, TOB1-AS1, and MALAT1 lncRNAs are sponges for miR-23b-3p, miR-27b-3p, and miR-125b-5p. Indeed, lncRNA knockdown significantly increased miR levels in U266 MM cells co-cultured with MM FB-EXOs. Selective miR-214-3p and miR-5100 overexpression modulates MAPK, PI3K/AKT/mTOR, and p53 pathways in MM cells. Interrogation using the DIANA tools algorithm and transient overexpression using miR mimic probes confirmed the involvement of miR-214-3p and miR-5100 and their target genes, PTEN and DUSP16, respectively, in the modulation of these intracellular pathways. Finally, the uptake of EXOs as well as miR-214-3p and miR-5100 overexpression increase MM cell proliferation and resistance to bortezomib-induced apoptosis by switching the balance between pro-/anti-apoptotic proteins. Overall, these data show that MM cells are not simply a container into which EXOs empty their cargo. On the contrary, tumour cells finely neutralize exosomal miRs via lncRNA expression to ensure their survival. © 2021 The Pathological Society of Great Britain and Ireland.
Assuntos
Exossomos , MicroRNAs , Mieloma Múltiplo , RNA Longo não Codificante , Exossomos/patologia , Fibroblastos/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mieloma Múltiplo/patologia , Fosfatidilinositol 3-Quinases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
OBJECTIVE: The aim of this study was to test the efficacy of autoantibodies to desmoglein 1 and desmoglein 3 detected by ELISA and indirect immunofluorescence in the diagnosis of oral pemphigus and to correlate the antibody titres with the severity of the disease. MATERIALS AND METHODS: We report a retrospective cohort study of 22 patients with oral pemphigus and 64 controls from a single tertiary centre. Data about histopathological examination, direct immunofluorescence, indirect immunofluorescence and ELISA were analysed. Global validation of ELISA and IIF both alone and combined was established by calculating sensitivity, specificity, accuracy and both positive predictive value and negative predictive value. The relationship between Oral Disease Severity Score values and ELISA titres was analysed using Pearson's coefficient. RESULTS: The best diagnostic performance was observed for anti-desmoglein 3 ELISA. The sensitivity was 75% and specificity 100% and positive predictive value and negative predictive value were 92.5% and accuracy 93.9%. The level of agreement with histopathology + direct immunofluorescence was substantial (k = .758). Anti-desmoglein 3 titres showed a significant correlation with Oral Disease Severity Score (p < .05). CONCLUSIONS: Serological tests are commonly employed during clinical practice as adjunctive tools. Anti-desmoglein 3 ELISA should be considered as a first-instance diagnostic test for oral pemphigus early detection.
Assuntos
Doenças da Boca , Úlceras Orais , Pênfigo , Estomatite , Autoanticorpos , Desmogleína 1 , Desmogleína 3 , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Pênfigo/patologia , Estudos RetrospectivosRESUMO
Ion channels are pore-forming proteins that allow ions to flow across plasma membranes and intracellular organelles in both excitable and non-excitable cells. They are involved in the regulation of several biological processes (i.e., proliferation, cell volume and shape, differentiation, migration, and apoptosis). Recently, the aberrant expression of ion channels has emerged as an important step of malignant transformation, tumor progression, and drug resistance, leading to the idea of "onco-channelopathy". Here, we review the contribution of ion channels and transporters in multiple myeloma (MM), a hematological neoplasia characterized by the expansion of tumor plasma cells (MM cells) in the bone marrow (BM). Deregulation of ion channels sustains MM progression by modulating intracellular pathways that promote MM cells' survival, proliferation, and drug resistance. Finally, we focus on the promising role of ion channels as therapeutic targets for the treatment of MM patients in a combination strategy with currently used anti-MM drugs to improve their cytotoxic activity and reduce adverse effects.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Humanos , Canais Iônicos/metabolismo , Íons/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismoRESUMO
Aberrant microRNA (miR) expression has an important role in tumour progression, but its involvement in bone marrow fibroblasts of multiple myeloma patients remains undefined. We demonstrate that a specific miR profile in bone marrow fibroblasts parallels the transition from monoclonal gammopathy of undetermined significance (MGUS) to myeloma. Overexpression of miR-27b-3p and miR-214-3p triggers proliferation and apoptosis resistance in myeloma fibroblasts via the FBXW7 and PTEN/AKT/GSK3 pathways, respectively. Transient transfection of miR-27b-3p and miR-214-3p inhibitors demonstrates a cooperation between these two miRNAs in the expression of the anti-apoptotic factor MCL1, suggesting that miR-27b-3p and miR-214-3p negatively regulate myeloma fibroblast apoptosis. Furthermore, myeloma cells modulate miR-27b-3p and miR-214-3p expression in fibroblasts through the release of exosomes. Indeed, tumour cell-derived exosomes induce an overexpression of both miRNAs in MGUS fibroblasts not through a simple transfer mechanism but by de novo synthesis triggered by the transfer of exosomal WWC2 protein that regulates the Hippo pathway. Increased levels of miR-27b-3p and miR-214-3p in MGUS fibroblasts co-cultured with myeloma cell-derived exosomes enhance the expression of fibroblast activation markers αSMA and FAP. These data show that the MGUS-to-myeloma transition entails an aberrant miRNA profile in marrow fibroblasts and highlight a key role of myeloma cells in modifying the bone marrow microenvironment by reprogramming the marrow fibroblasts' behaviour. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Células da Medula Óssea/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , MicroRNAs/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Mieloma Múltiplo/metabolismo , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Células da Medula Óssea/patologia , Células Cultivadas , Progressão da Doença , Endopeptidases , Exossomos/genética , Exossomos/patologia , Proteína 7 com Repetições F-Box-WD/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Feminino , Fibroblastos/patologia , Gelatinases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Microambiente Tumoral , Regulação para CimaRESUMO
MicroRNAs (miRNAs, or miRs) are single-strand short non-coding RNAs with a pivotal role in the regulation of physiological- or disease-associated cellular processes. They bind to target miRs modulating gene expression at post-transcriptional levels. Here, we present an overview of miRs deregulation in the pathogenesis of multiple myeloma (MM), and discuss the potential use of miRs/nanocarriers association in clinic. Since miRs can act as oncogenes or tumor suppressors, strategies based on their inhibition and/or replacement represent the new opportunities in cancer therapy. The miRs delivery systems include liposomes, polymers, and exosomes that increase their physical stability and prevent nuclease degradation. Phase I/II clinical trials support the importance of miRs as an innovative therapeutic approach in nanomedicine to prevent cancer progression and drug resistance. Results in clinical practice are promising.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Transferência de Genes , MicroRNAs/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Nanotecnologia , Terapêutica com RNAi , Animais , Progressão da Doença , Exossomos , Regulação Neoplásica da Expressão Gênica , Humanos , Lipídeos/química , Lipossomos , Mieloma Múltiplo/patologia , Nanotecnologia/métodos , Polímeros/química , Terapêutica com RNAi/métodosRESUMO
BACKGROUND: Multiple myeloma (MM) is a cancer of terminally differentiated plasma that is part of a spectrum of blood diseases. The role of the micro-environment is crucial for MM clonal evolution. METHODS: This paper describes the analysis carried out on a limited number of genes automatically extracted by a nonnegative matrix factorization (NMF) based approach from gene expression profiles of bone marrow fibroblasts of patients with monoclonal gammopathy of undetermined significance (MGUS) and MM. RESULTS: Automatic exploration through NMF, combined with a motivated post-processing procedure and a pathways analysis of extracted genes, allowed to infer that a functional switch is required to lead fibroblasts to acquire pro-tumorigenic activity in the progression of the disease from MGUS to MM. CONCLUSION: The extracted biologically relevant genes may be representative of the considered clinical conditions and may contribute to a deeper understanding of tumor behavior.
Assuntos
Algoritmos , Células da Medula Óssea/patologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.
Assuntos
Fibroblastos/efeitos dos fármacos , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/patologia , Actinas/biossíntese , Actinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/genética , Método Duplo-Cego , Endopeptidases , Epoetina alfa , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatinases/biossíntese , Gelatinases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neovascularização Fisiológica/efeitos dos fármacos , Receptores da Eritropoetina/biossíntese , Receptores da Eritropoetina/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genéticaRESUMO
Many researchers have speculated that the clinical progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) is driven by defects in dendritic cell (DC) function. However, evidence supporting this assumption is controversial, and no mechanism for the putative DC dysfunction has been demonstrated thus far. We studied DC subsets from the bone marrow of MM patients compared with those of MGUS patients and control subjects. We found that myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) accumulate in the bone marrow during the MGUS-to-MM progression. After engulfment of apoptotic tumor plasma cells via CD91, bone marrow mDCs and pDCs mature and are able to activate tumor-specific CD8(+) T cells. However, by interacting directly with CD28 on live (nonapoptotic) tumor plasma cells, bone marrow mDCs downregulate the expression of proteasome subunits in these cells, thus enabling their evasion from human leukocyte antigen (HLA) class I-restricted CD8(+) T-cell killing. These results suggest that DCs play a dual, but opposing, role in MM: for one, DCs activate CD8(+) T cells against tumor plasma cells and, for the other, DCs protect tumor plasma cells from CD8(+) T-cell killing. This information should be taken into account in designing immunotherapy approaches to enhance immune surveillance in MGUS and to break down immune tolerance in MM.
Assuntos
Medula Óssea/patologia , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/imunologia , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Plasmocitoma/patologia , Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular , Células Cultivadas , Células Dendríticas/patologia , Humanos , Mieloma Múltiplo/imunologia , Plasmócitos/imunologia , Plasmocitoma/imunologia , Células Tumorais CultivadasRESUMO
The reinforcement of the defense mechanism of fish, through the administration of immunostimulants, is considered as a promising alternative to vaccines. Natural immunostimulants such as polyphenols, flavanoids, pigments and essential oils can modulate the innate immune response. In lower vertebrates, melano-macrophage centres, i.e. clusters of pigment-containing cells forming the extracutaneous pigment system, are wide-spread in the stroma of the haemopoietic tissue, mainly in kidney and spleen. In fishes, melano-macrophage centres play an important role in the immune response against antigenic stimulants and pathogens. In the present study, we evaluated the effect of a polyphenol-enriched diet on the health status of European sea bass (Dicentrarchus labrax L.). Farmed sea bass were administered a feed containing a phytocomplex, rich in catechins and epigallocatechins, which was obtained from the seeds of Canosina Nero di Troia Vitis vinifera and mixed with conventional feed at two different concentrations. The effects of such a diet were investigated in juvenile and commercial size samples, i.e. undergoing a short- and long-term period of diet, respectively, focusing on their extracutaneous pigmentary system and, in more detail, on the enzymatic activities leading to melanin biosynthesis. Our results show that prolonged dietary treatments with higher concentration of polyphenols might modulate tyrosinase activity and gene expression in commercial size fishes. An increase of melano-macrophage activity is correlated to a stimulation of cytoprotective functions against antigenic stimulants and pathogens, as an expression of a robust and protective adaptive immune response.
Assuntos
Bass/imunologia , Dieta/veterinária , Imunidade Inata/efeitos dos fármacos , Macrófagos/imunologia , Polifenóis/farmacologia , Ração Animal/análise , Animais , Suplementos Nutricionais/análise , Rim/efeitos dos fármacos , Rim/imunologia , Macrófagos/efeitos dos fármacos , Polifenóis/administração & dosagemRESUMO
Multiple myeloma (MM) pathogenesis and progression largely rely on the cells and extracellular factors in the bone marrow (BM) microenvironment. Compelling studies have identified tumour exosomes as key regulators in the maintenance and education of the BM microenvironment by targeting stromal cells, immune cells, and vascular cells. However, the role of MM exosomes in the modification of the BM microenvironment and MM progression remains unclear. Here, we explored the functions of MM exosomes in angiogenesis and immunosuppression in vitro and in vivo. Murine MM exosomes carrying multiple angiogenesis-related proteins enhanced angiogenesis and directly promoted endothelial cell growth. Several pathways such as signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase, and p53 were modulated by the exosomes in endothelial and BM stromal cells. These exosomes promoted the growth of myeloid-derived suppressor cells (MDSCs) in naive mice through activation of the STAT3 pathway and changed their subsets to similar phenotypes to those seen in MM-bearing mice. Moreover, MM exosomes up-regulated inducible nitric oxide synthase and enhanced the immunosuppressive capacity of BM MDSCs in vivo. Our data show that MM exosomes modulate the BM microenvironment through enhancement of angiogenesis and immunosuppression, which will further facilitate MM progression. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Microambiente Celular , Exossomos/imunologia , Tolerância Imunológica , Mieloma Múltiplo/imunologia , Neovascularização Patológica , Animais , Medula Óssea/imunologia , Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Exossomos/patologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/irrigação sanguínea , Mieloma Múltiplo/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and ß-dystroglycan (αDG, ßDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier control occurring in the adult dystrophic brain.
Assuntos
Separação Celular/métodos , Distrofia Muscular Animal/patologia , Células-Tronco Neurais/citologia , Antígeno AC133/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Aquaporina 4/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Distroglicanas/metabolismo , Distrofina/metabolismo , Citometria de Fluxo , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Membrana , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Proteínas Musculares , Distrofia Muscular Animal/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/ultraestrutura , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esferoides Celulares/citologia , Esferoides Celulares/ultraestrutura , Ubiquitina/metabolismoRESUMO
Regulatory T cells (Tregs) are essential for maintenance of self-tolerance; however, tumor cells can exploit the tolerance to escape the immune system. We investigated the Tregs frequency in patients with multiple myeloma (MM) and in those with monoclonal gammopathy of undetermined significance (MGUS), and found that CD4(+) FoxP3(+) and CD8(+) FoxP3(+) Tregs were significantly increased in patients with MM and correlated with the active phase. Both Tregs subsets were expanded in cocultures of CD3(+) lymphocytes and fresh CD138(+) MM plasma cells or RPMI8226 and U266 cell lines and functioned as natural (n) and inducible (i) Tregs insofar as they inhibited the proliferation of stimulated CD3 lymphocytes via contact-dependent and contact-independent pathways. Induction of Tregs by MM plasma cells required a contact-dependent pathway, implying antigen recognition by T cells. MM plasma cells acted as immature and tolerogenic antigen-presenting cells (APCs), in that they displayed low CD80/CD86 expression associated with a phagocytic activity. By acting as immature APCs, MM plasma cells plausibly expand (n)Tregs and (i)Tregs both through conversion of CD3(+) FoxP3(-) into CD3(+) FoxP3(+) T cells and proliferation of CD3(+) FoxP3(+) T cells, which may suppress the anti-MM immune response.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Regulação Neoplásica da Expressão Gênica , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Mieloma Múltiplo/imunologia , Plasmócitos/imunologia , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/patologia , Complexo CD3/genética , Complexo CD3/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Comunicação Celular , Proliferação de Células , Técnicas de Cocultura , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Humanos , Tolerância Imunológica , Imunofenotipagem , Ativação Linfocitária , Gamopatia Monoclonal de Significância Indeterminada/genética , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Transdução de Sinais , Sindecana-1/genética , Sindecana-1/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Tumour pathogenesis in multiple myeloma (MM) correlates with a high vascular index. Therefore, targeting angiogenesis is an important therapeutic tool to reduce MM progression. This study aimed to investigate the role of invariant natural killer T (iNKT) cells in angiogenesis and the mechanisms behind the stimulation by α-Galactosylceramide (α-GalCer). We have previously found that α-GalCer could increase the survival of 5T33MM mice and here we demonstrate that α-GalCer reduces the microvessel density. We performed both in vivo and in vitro angiogenic assays to confirm this observation. We found that conditioned medium of α-GalCer stimulated iNKT cells reduced neovascularization in the chick chorioallantoic membrane and in matrigel plug assays. Moreover, we observed a reduction in proliferation, migration and network formation and an induction of apoptosis upon exposure of murine endothelial cell lines to this conditioned medium. We furthermore observed that the JAK-STAT signaling pathway was highly activated in endothelial cells in response to stimulated iNKT cells, indicating the possible role of IFN-γ in the anti-angiogenic process. In conclusion, these results highlight the possibility of recruiting iNKT cells to target MM and angiogenesis. This gives a rationale for combining immunotherapy with conventional anti-tumour treatments in view of increasing their therapeutic potential.
Assuntos
Galactosilceramidas/farmacologia , Janus Quinases/imunologia , Mieloma Múltiplo/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Fatores de Transcrição STAT/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Imunoterapia/métodos , Interferon gama/imunologia , Camundongos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Células T Matadoras Naturais/patologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Transdução de Sinais/imunologiaRESUMO
Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that binds with high affinity and selectivity to fibroblast growth factor-2 (FGF2), thus inhibiting its pro-angiogenic activity. Here we investigated the effects of PTX3 on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patient-derived bone marrow (BM) plasma cells (PCs), endothelial cells (ECs), and fibroblasts (FBs), and assessed whether PTX3 can modulate the cross-talk between PCs and those microenvironment cells. PTX3 and FGF2 expression was evaluated by ELISA. Functional studies, including cell viability, wound healing, chemotaxis, and Matrigel(®) assays, were performed on MGUS and MM ECs and FBs upon the PTX3 treatment. Through western blot PTX3-induced modulation in FGF2/FGF receptor signalling pathways was evaluated in MGUS and MM ECs and FBs through western blot. Co-cultures between MM ECs/FBs and human PC lines were used to evaluate possible PTX3 indirect effects on MM PCs. Adhesion molecules were studied by flow cytometry. PTX3 provides a direct time- and dose-dependent apoptotic effect on MM ECs and FBs, but not on either MM primary PCs or human PC lines. PTX3 inhibits migration of MM ECs and FBs in a dose-dependent manner, and impacts in vitro and in vivo FGF2-mediated MM angiogenesis. Co-cultures of PCs and ECs/FBs show that PTX3 treatment indirectly impairs PC viability and adhesion. We conclude that PTX3 is an anti-angiogenic factor in MM and behaves as a cytotoxic molecule on MM cells by inhibiting the cross-talk between PCs and ECs/FBs.
Assuntos
Células da Medula Óssea/metabolismo , Proteína C-Reativa/metabolismo , Comunicação Celular , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Mieloma Múltiplo/metabolismo , Plasmócitos/metabolismo , Componente Amiloide P Sérico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Western Blotting , Células da Medula Óssea/patologia , Estudos de Casos e Controles , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Microambiente Celular , Quimiotaxia , Embrião de Galinha , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/patologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/metabolismo , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/irrigação sanguínea , Mieloma Múltiplo/patologia , Neovascularização Patológica , Plasmócitos/patologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM niche, MM cells adhere to FBs sustaining immune evasion, drug resistance and the undetectable endurance of tumor cells known as minimal residual disease (MRD). Here, we describe the novel bi-specific designed ankyrin repeat protein (DARPin) α-FAPx4-1BB (MP0310) with FAP-dependent 4-1BB agonistic activity. The α-FAPx4-1BB DARPin simultaneously binds to FAP and 4-1BB overexpressed by activated FBs and immune cells, respectively. Although flow cytometry analysis showed that T and NK cells from MM patients were not activated and did not express 4-1BB, stimulation with daratumumab or elotuzumab, monoclonal antibodies (mAbs) currently used for the treatment of MM, significantly upregulated 4-1BB both in vitro and in MM patients following mAb-based therapy. The mAb-induced 4-1BB overexpression allowed the engagement of α-FAPx4-1BB that acted as a bridge between FAP+FBs and 4-1BB+NK cells. Therefore, α-FAPx4-1BB enhanced both the adhesion of daratumumab-treated NK cells on FBs as well as their activation by improving release of CD107a and perforin, hence MM cell killing via antibody-mediated cell cytotoxicity (ADCC). Interestingly, α-FAPx4-1BB significantly potentiated daratumumab-mediated ADCC in the presence of FBs, suggesting that it may overcome the BM FBs' immunosuppressive effect. Overall, we speculate that treatment with α-FAPx4-1BB may represent a valuable strategy to improve mAb-induced NK cell activity fostering MRD negativity in MM patients through the eradication of latent MRD cells.
Assuntos
Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais , Células Matadoras Naturais , Mieloma Múltiplo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Humanos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/agonistas , EndopeptidasesRESUMO
Angiogenesis represents a pivotal hallmark of multiple myeloma (MM) that correlates to patients' prognosis, overall survival, and drug resistance. Hence, several anti-angiogenic drugs that directly target angiogenic cytokines (i.e., monoclonal antibodies, recombinant molecules) or their cognate receptors (i.e., tyrosine kinase inhibitors) have been developed. Additionally, many standard antimyeloma drugs currently used in clinical practice (i.e., immunomodulatory drugs, bisphosphonates, proteasome inhibitors, alkylating agents, glucocorticoids) show anti-angiogenic effects further supporting the importance of inhibiting angiogenesis from potentiating the antimyeloma activity. Here, we review the most important anti-angiogenic therapies used for the management of MM patients with a particular focus on their pharmacological profile and on their anti-angiogenic effect in vitro and in vivo. Despite the promising perspective, the direct targeting of angiogenic cytokines/receptors did not show a great efficacy in MM patients, suggesting the need to a deeper knowledge of the BM angiogenic niche for the design of novel multi-targeting anti-angiogenic therapies.
RESUMO
Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication within the bone marrow (BM) of multiple myeloma (MM) patients, where they mediate several tumor-associated processes. Here, we investigate the contribution of fibroblasts-derived EVs (FBEVs) in supporting BM angiogenesis. We demonstrate that FBEVs' cargo contains several angiogenic cytokines (i.e., VEGF, HGF, and ANG-1) that promote an early over-angiogenic effect independent from EVs uptake. Interestingly, co-culture of endothelial cells from MM patients (MMECs) with FBEVs for 1 or 6 h activates the VEGF/VEGFR2, HGF/HGFR, and ANG-1/Tie2 axis, as well as the mTORC2 and Wnt/ß-catenin pathways, suggesting that the early over-angiogenic effect is a cytokine-mediated process. FBEVs internalization occurs after longer exposure of MMECs to FBEVs (24 h) and induces a late over-angiogenic effect by increasing MMECs migration, chemotaxis, metalloproteases release, and capillarogenesis. FBEVs uptake activates mTORC1, MAPK, SRC, and STAT pathways that promote the release of pro-angiogenic cytokines, further supporting the pro-angiogenic milieu. Overall, our results demonstrate that FBEVs foster MM angiogenesis through dual time-related uptake-independent and uptake-dependent mechanisms that activate different intracellular pathways and transcriptional programs, providing the rationale for designing novel anti-angiogenic strategies.
RESUMO
We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8(+) T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8(+) T cells lysed autologous transformed plasma cells more than MM CD8(+) T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with delta, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.