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1.
Blood Cancer J ; 14(1): 75, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38697976

RESUMO

Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.


Assuntos
Galectinas , Linfonodos , Linfoma Folicular , Microambiente Tumoral , Humanos , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Linfoma Folicular/terapia , Linfonodos/patologia , Linfonodos/imunologia , Microambiente Tumoral/imunologia , Esferoides Celulares , Imunoterapia/métodos , Transdução de Sinais , Células Tumorais Cultivadas
2.
J Immunother Cancer ; 11(10)2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37899130

RESUMO

BACKGROUND: Follicular lymphoma (FL), the most common indolent non-Hodgkin's Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses. METHODS: To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability. RESULTS: FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3. CONCLUSIONS: In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.


Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/genética , Receptor Celular 2 do Vírus da Hepatite A , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral , Medicina de Precisão
3.
Leukemia ; 37(6): 1311-1323, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37031299

RESUMO

Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.


Assuntos
Linfoma de Célula do Manto , Humanos , Adulto , Linhagem Celular Tumoral , Linfoma de Célula do Manto/patologia , Resistencia a Medicamentos Antineoplásicos , Adenina/uso terapêutico , Microambiente Tumoral
4.
J Immunother Cancer ; 11(2)2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36854569

RESUMO

BACKGROUND: Approximately one-third of diffuse large B cell lymphoma (DLBCL) patients exhibit co-expression of MYC and BCL2 (double-expressor lymphoma, DEL) and have a dismal prognosis. Targeted inhibition of the anti-apoptotic protein BCL2 with venetoclax (ABT-199) has been approved in multiple B-cell malignancies and is currently being investigated in clinical trials for DLBCL. Whether BCL2 anti-apoptotic function represents a multifaceted vulnerability for DEL-DLBCL, affecting both lymphoma B cells and T cells within the tumor microenvironment, remains to be elucidated. METHODS: Here, we present novel genetically engineered mice that preclinically recapitulate DEL-DLBCL lymphomagenesis, and evaluate their sensitivity ex vivo and in vivo to the promising combination of venetoclax with anti-CD20-based standard immunotherapy. RESULTS: Venetoclax treatment demonstrated specific killing of MYC+/BCL2+ lymphoma cells by licensing their intrinsically primed apoptosis, and showed previously unrecognized immunomodulatory activity by specifically enriching antigen-activated effector CD8 T cells infiltrating the tumors. Whereas DEL-DLBCL mice were refractory to venetoclax alone, inhibition of BCL2 significantly extended overall survival of mice that were simultaneously treated with a murine surrogate for anti-CD20 rituximab. CONCLUSIONS: These results suggest that the combination of anti-CD20-based immunotherapy and BCL2 inhibition leads to cooperative immunomodulatory effects and improved preclinical responses, which may offer promising therapeutic opportunities for DEL-DLBCL patients.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Imunoterapia , Linfoma Difuso de Grandes Células B , Animais , Camundongos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Modelos Animais de Doenças , Imunoterapia/métodos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2 , Microambiente Tumoral , Proteínas Proto-Oncogênicas c-myc
5.
FEBS J ; 289(9): 2540-2561, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34796659

RESUMO

Cardiovascular diseases are the leading cause of death globally and more than four out of five cases are due to ischemic events. Cardiac fibroblasts (CF) contribute to normal heart development and function, and produce the post-ischemic scar. Here, we characterize the biochemical and functional aspects related to CF endurance to ischemia-like conditions. Expression data mining showed that cultured human CF (HCF) express more BCL2 than pulmonary and dermal fibroblasts. In addition, gene set enrichment analysis showed overrepresentation of genes involved in the response to hypoxia and oxidative stress, respiration and Janus kinase (JAK)/Signal transducer and Activator of Transcription (STAT) signaling pathways in HCF. BCL2 sustained survival and proliferation of cultured rat CF, which also had higher respiration capacity and reactive oxygen species (ROS) production than pulmonary and dermal fibroblasts. This was associated with higher expression of the electron transport chain (ETC) and antioxidant enzymes. CF had high phosphorylation of JAK2 and its effectors STAT3 and STAT5, and their inhibition reduced viability and respiration, impaired ROS control and reduced the expression of BCL2, ETC complexes and antioxidant enzymes. Together, our results identify molecular and biochemical mechanisms conferring survival advantage to experimental ischemia in CF and show their control by the JAK2/STAT signaling pathway. The presented data point to potential targets for the regulation of cardiac fibrosis and also open the possibility of a general mechanism by which somatic cells required to acutely respond to ischemia are constitutively adapted to survive it.


Assuntos
Antioxidantes , Janus Quinase 2 , Animais , Fibroblastos/metabolismo , Isquemia , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Respiração , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
6.
Leukemia ; 35(9): 2635-2649, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33731849

RESUMO

Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures (n = 19) and in vivo mouse co-xenografts demonstrated that FL-FDC crosstalk favors tumor growth and, via the secretion of CCL2 and CSF-1, promotes monocyte recruitment, differentiation, and polarization towards an M2-like protumoral phenotype. Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Folicular/patologia , Macrófagos/patologia , Monócitos/patologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Microambiente Tumoral , Aminopiridinas/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Diferenciação Celular , Proliferação de Células , Humanos , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Pirróis/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS One ; 9(10): e110979, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25330150

RESUMO

BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.


Assuntos
Antineoplásicos/efeitos adversos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Cisplatino/efeitos adversos , Perda Auditiva , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Nefropatias/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Morte Celular/efeitos dos fármacos , Cisplatino/farmacologia , Modelos Animais de Doenças , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/patologia , Masculino , Camundongos , Ratos , Traumatismo por Reperfusão/patologia , Proteínas de Peixe-Zebra/metabolismo
8.
PLoS One ; 7(6): e38620, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22745672

RESUMO

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.


Assuntos
Calpaína/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Microscopia Confocal , Antígenos de Histocompatibilidade Menor , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética
9.
J Cell Sci ; 124(Pt 4): 556-64, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21245196

RESUMO

Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived 'poropeptide' produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.


Assuntos
Antineoplásicos/metabolismo , Apoptose , Neoplasias/fisiopatologia , Peptídeos/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citocromos c/metabolismo , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Peptídeos/química , Peptídeos/genética , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/farmacologia
10.
PLoS One ; 5(2): e9066, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-20140092

RESUMO

BACKGROUND: The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. CONCLUSION/SIGNIFICANCE: BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.


Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Lipídeos de Membrana/química , Fragmentos de Peptídeos/química , Proteína X Associada a bcl-2/química , Proteína bcl-X/química , Sequência de Aminoácidos , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , Citocromos c/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
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