Toll-like receptor 4 selective inhibition in medullar microenvironment alters multiple myeloma cell growth.

peer reviewedBone marrow (BM) mesenchymal stromal cells (MSCs) are abnormal in multiple myeloma (MM) and play a critical role by promoting growth, survival, and drug resistance of MM cells. We observed higher Toll-like receptor 4 (TLR4) gene expression in MM MSCs than in MSCs from healthy donors. At the clinical level, we highlighted that TLR4 expression in MM MSCs evolves in parallel with the disease stage. Thus, we reasoned that the TLR4 axis is pivotal in MM by increasing the protumor activity of MSCs. Challenging primary MSCs with TLR4 agonists increased the expression of CD54 and interleukin-6 (IL-6), 2 factors directly implicated in MM MSC-MM cell crosstalk. Then, we evaluated the therapeutic efficacy of a TLR4 antagonist combined or not with conventional treatment in vitro with MSC-MM cell coculture and in vivo with the Vk*MYC mouse model. Selective inhibition of TLR4 specifically reduced the MM MSC ability to support the growth of MM cells in an IL-6-dependent manner and delayed the development of MM in the Vk*MYC mouse model by altering the early disease phase in vivo. For the first time, we demonstrate that specific targeting of the pathological BM microenvironment via TLR4 signaling could be an innovative approach to alter MM pathology development

Three-dimensional modeling of human bone marrow and skin tissue for the exploration of haematopoietic cells in skin repair

Les défauts de cicatrisation des plaies cutanées touchent des millions de personnes âgées ou souffrant de diverses pathologies telles que le diabète et l’obésité. Ces défauts sont causés en partie par la dérégulation de l’hématopoïèse, impliquant une production mal régulée par la moelle osseuse (MO) de cellules myéloïdes qui infiltrent ensuite la plaie cutanée. L’hématopoïèse et les interactions entre la MO et la peau sont des processus complexes qui n'ont pas encore été entièrement élucidés chez l’Homme, principalement en raison des limites des modèles de culture in vitro existants et des modèles animaux imparfaits. Dans ce projet, des modèles de MO humaine et de peau tridimensionnel ont été développés pour améliorer l'étude du rôle de l’hématopoïèse après une lésion cutanée. Un protocole de production de cellules hématopoïétiques in vitro a d’abord été établi. En utilisant un procédé de culture en deux phases, un stroma médullaire complexe a été généré en utilisant des cellules primaires humaines récupérées à partir d'échantillons de MO. Ce stroma comprend les composants cellulaires non hématopoïétiques essentiels des niches hématopoïétiques de la MO, notamment les cellules stromales mésenchymateuses (CSM), les ostéoblastes, les adipocytes et des cellules endothéliales organisées en réseau. Il exprime des facteurs pro-hématopoïétiques essentiels pour la fonctionnalité des différentes niches de la MO. Par la suite, des cellules CD34+, isolées du sang de cordon ombilical ou de la MO, ont été utilisées dans des expériences de co-culture. Les cellules hématopoïétiques ont été analysées par cytométrie en flux après 14, 21 et 28 jours de co-culture. Les résultats ont montré que le stroma préalablement préparé, était capable de soutenir les cellules souches et progénitrices hématopoïétiques (CSPH), de diriger la différenciation hématopoïétique et de produire des cellules myéloïdes sans ajout de cytokines exogènes. De plus, ce modèle a été capable de s'adapter à un défi inflammatoire en ré-orientant l'hématopoïèse. En outre, une preuve de concept a été réalisée avec succès, montrant la possibilité d’étendre l'approche de modélisation de la MO à un contexte tridimensionnel en sphéroïde. Simultanément, un modèle de peau humaine tridimensionnel a été développé. Ce modèle a été construit par auto-organisation de cellules primaires issues de biopsies cutanées en sphéroïdes, qui ont servi d’unités de base de construction. Ces sphéroïdes ont été couplées avec des polymères naturels et/ou synthétiques et mis en maturation en utilisant une interface air-liquide pendant trois semaines, pour simuler la structure de la peau. Les analyses histologiques et en immunofluorescence ont révélé une organisation structurée du tissu avec des couches dermique et épidermique distinctes, une pigmentation ainsi qu'une grande complexité cellulaire, incluant une pré-vascularisation, des cellules immunitaires et des glandes sudoripares, indiquant un vrai mimétisme du modèle avec la peau native humaine. La perspective d’intégration de ces deux modèles humains dans un même système ouvre de nouvelles voies pour l'investigation du rôle de l’hématopoïèse dans la réparation cutanée ainsi que pour tester et mettre au point des thérapies innovantes modulant l’inflammation lors de défauts de réparation cutanée.The healing defects of cutaneous wounds affect millions of elderly individuals or suffering from various pathologies such as diabetes and obesity. These defects are partly caused by the deregulation of hematopoiesis, involving poorly regulated production by the bone marrow (BM) of myeloid cells that subsequently infiltrate the cutaneous wound. Haematopoiesis and BM-skin crosstalk are complex processes not completely elucidated yet, mainly due to limits of existing in vitro culture models and imperfect animal models. In this project, human BM and three-dimensional skin models have been developed to enhance the study of the hematopoiesis role after skin injury. At first, a protocol for in vitro production of hematopoietic cells was established. Through a two-phase culture process, a rich medullary stroma was generated using primary human cells from BM samples. This stroma included the essential non-hematopoietic cellular components of BM hematopoietic niches including mesenchymal stromal cells (MSC), osteoblasts, adipocytes and endothelial cells organized into a network. It expressed essential pro-hematopoietic factors for the functionality of the various BM niches. Subsequently, CD34+ cells isolated from umbilical cord blood or BM, were used in co-culture experiments with medullary stroma. Hematopoietic cells were analyzed using flow cytometry after 14, 21, and 28 days of co-culture. Results showed that stroma was able to sustain hematopoietic stem and progenitor cells (HSPC), to drive hematopoietic differentiation and to produce myeloid cells without exogenous cytokine addition. Furthermore, this model was able to adapt to an inflammatory challenge by modulating the direction of hematopoiesis. In addition, a proof of concept was successfully demonstrated, showing the possibility of extending the BM modeling approach to a three-dimensional spheroid context. Simultaneously, a 3D human skin model was developed. This model was constructed by self-organization of primary cells from skin biopsies into spheroids, which served as building blocks. These spheroids were then merged with natural and/ or synthetic polymers and matured at an air-liquid interface for three weeks to simulate skin structure. Histological and immunofluorescence analyses revealed a structured organization of the tissue with distinct dermal and epidermal layers, pigmentation, and high cellular complexity, including pre-vascularization, immune cells, and sweat glands, indicating a true mimicry of the model with native human skin. The prospect of integrating these two human models into a single system opens new pathways for investigating the role of hematopoiesis in cutaneous repair as well as for testing and developing innovative therapies modulating inflammation during cutaneous repair defects

: MULTIPLE MYELOMA MSC ARE ABNORMAL

International audienceRecent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells

Functional Comparison between Healthy and Multiple Myeloma Adipose Stromal Cells

Multiple myeloma (MM) is an incurable B cell neoplasia characterized by the accumulation of tumor plasma cells within the bone marrow (BM). As a consequence, bone osteolytic lesions develop in 80% of patients and remain even after complete disease remission. We and others had demonstrated that BM-derived mesenchymal stromal cells (MSCs) are abnormal in MM and thus cannot be used for autologous treatment to repair bone damage. Adipose stromal cells (ASCs) represent an interesting alternative to MSCs for cellular therapy. Thus, in this study, we wondered whether they could be a good candidate in repairing MM bone lesions. For the first time, we present a transcriptomic, phenotypic, and functional comparison of ASCs from MM patients and healthy donors (HDs) relying on their autologous MSC counterparts. In contrast to MM MSCs, MM ASCs did not exhibit major abnormalities. However, the changes observed in MM ASCs and the supportive property of ASCs on MM cells question their putative and safety uses at an autologous or allogenic level

Integrated Transcriptomic, Phenotypic, and Functional Study Reveals Tissue-Specific Immune Properties of Mesenchymal Stromal Cells

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