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1.
Am J Hematol ; 97(9): 1200-1214, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35759575

RESUMO

Human endogenous retroviruses (HERVs) represent 8% of the human genome. The expression of HERVs and their immune impact have not been extensively studied in Acute Myeloid Leukemia (AML). In this study, we used a reference of 14 968 HERV functional units to provide a thorough analysis of HERV expression in normal and AML bone marrow cells. We show that the HERV retrotranscriptome accurately characterizes normal and leukemic cell subpopulations, including leukemia stem cells, in line with different epigenetic profiles. We then show that HERV expression delineates AML subtypes with different prognoses. We finally propose a method to select and prioritize CD8+ T cell epitopes derived from AML-specific HERVs and we show that lymphocytes infiltrating patient bone marrow at diagnosis contain naturally occurring CD8+ T cells against these HERV epitopes. We also provide in vitro data supporting the functionality of HERV-specific CD8+ T-cells against AML cells. These results show that HERVs represent an important source of genetic information that can help enhancing disease stratification or biomarker identification and an important reservoir of alternative tumor-specific T cell epitopes relevant for cancer immunotherapy.


Assuntos
Retrovirus Endógenos , Leucemia Mieloide Aguda , Linfócitos T CD8-Positivos , Retrovirus Endógenos/genética , Epitopos de Linfócito T , Humanos , Imunoterapia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Células-Tronco
2.
J Vis Exp ; (190)2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36591983

RESUMO

The medullary niche is a complex ecosystem that is essential to maintain homeostasis for resident cells. Indeed, the bone marrow, which includes a complex extracellular matrix and various cell types, such as mesenchymal stem cells, osteoblasts, and endothelial cells, is deeply involved in hematopoietic stem cell regulation through direct cell-cell interactions, as well as cytokine production. To closely mimic this in vivo structure and conduct experiments reflecting the responses of the human bone marrow, several 3D models have been created based on biomaterials, relying primarily on primary stromal cells. Here, a protocol is described to obtain a minimal and standardized system that is easy to set up and provides features of bone marrow-like structure, which combines different cell populations including endothelial cells, and reflects the heterogeneity of in vivo bone marrow tissue. This 3D bone marrow-like structure-assembled using calcium phosphate-based particles and human cell lines, representative of the bone marrow microenvironment-allows the monitoring of a wide variety of biological processes by combining or replacing different primary cell populations within the system. The final 3D structures can then either be harvested for image analysis after fixation, paraffin-embedding, and histological/immunohistochemical staining for cell localization within the system, or dissociated to collect each cellular component for molecular or functional characterization.


Assuntos
Medula Óssea , Neoplasias , Humanos , Medula Óssea/fisiologia , Células Endoteliais , Ecossistema , Células-Tronco Hematopoéticas , Células Estromais , Células da Medula Óssea , Neoplasias/metabolismo , Nicho de Células-Tronco/fisiologia , Microambiente Tumoral
3.
Biomater Sci ; 10(2): 485-498, 2022 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-34904143

RESUMO

Bone marrow is a complex and dynamic microenvironment that provides essential cues to resident cells. We developed a standardized three-dimensional (3D) model to decipher mechanisms that control human cells during hematological and non-hematological processes. Our simple 3D-model is constituted of a biphasic calcium phosphate-based scaffold and human cell lines to ensure a high reproducibility. We obtained a minimal well-organized bone marrow-like structure in which various cell types and secreted extracellular matrix can be observed and characterized by in situ imaging or following viable cell retrieval. The complexity of the system can be increased and customized, with each cellular component being independently modulated according to the issue investigated. Introduction of pathological elements in this 3D-system accurately reproduced changes observed in patient bone marrow. Hence, we have developed a handy and flexible standardized microphysiological system that mimics human bone marrow, allowing histological analysis and functional assays on collected cells.


Assuntos
Medula Óssea , Osso e Ossos , Células da Medula Óssea , Matriz Extracelular , Humanos , Reprodutibilidade dos Testes
4.
Haematologica ; 106(1): 111-122, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32001529

RESUMO

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Células-Tronco Neoplásicas , Proteína Morfogenética Óssea 4 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases , Fator de Transcrição STAT3/genética , Microambiente Tumoral
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