New insights into the role of glycosaminoglycans in the endosteal bone microenvironment

The bone microenvironment is a complex tissue in which heterogeneous cell populations of hematopoietic and mesenchymal origin interact with environmental cues to maintain tissue integrity. Both cellular and matrix components are subject to physiologic challenges and can dynamically respond by modifying cell/matrix interactions. When either component is impaired, the physiologic balance is lost. Here, we review the current state of knowledge of how glycosaminoglycans – organic components of the bone extracellular matrix – influence the bone micromilieu. We point out how they interact with mediators of distinct signaling pathways such as the RANKL/OPG axis, BMP and WNT signaling, and affect the activity of bone remodeling cells within the endosteal niche summarizing their potential for therapeutic intervention.

Morphogens and growth factor signalling in the myeloma bone-lining niche

Multiple myeloma is a malignancy caused by the clonal expansion of abnormal plasma cells. Myeloma cells have proven to be incredibly successful at manipulating their microenvironment to promote growth and to evade modern therapies. They have evolved to utilise the integral signalling pathways of the bone and bone marrow to drive disease progression. The bone marrow is often described in the context of a single structure that fills the bone cavity and supports normal haematopoiesis. However, within that structure exists two anatomically different niches, the perivascular niche and the endosteal niche. These contain different cell types functioning to support normal immune and blood cell production as well as healthy bone. These cells secrete numerous signalling molecules that can influence myeloma cell biology and behaviour. The endosteal niche is home to specific bone cell lineages and plays a pivotal role in myeloma cell establishment and survival. This review will concentrate on some of the signalling pathways that are hijacked by myeloma cells to shape a favourable environment, and the different influences myeloma cells are exposed to depending on their spatial location within the bone marrow.

Levels of Osteopontin (SPP1), Osteonectin (SPARC) and Biglycan (BGN) in Acute Myeloid Leukemia Bone Marrow Biopsies Post-Induction Therapy Define the Status of Osteogenic Niche and Show Inverse Correlation with Therapeutic Response

Background: Acute Myeloid Leukemia (AML) has a five-year survival rate of 25% and its high mortality is linked to poor response to treatment and relapse. Our understanding of the molecular mechanisms controlling relapse and AML progression is limited. Animal models indicate that AML cells significantly modulate their bone marrow microenvironment inducing gradual loss of endosteal and vascular niches, both playing critical roles in support and maintenance of normal hematopoiesis. The goal of this study was to determine microenvironmental factors driving the gradual retraction of endosteal and vascular niches directly in the AML core bone marrow biopsies, and assess the treatment effect on hematopoietic and non-hematopoietic cells. Methods: Transcriptomics and histopathologic evaluations of matched human AML core bone marrow biopsies obtained at diagnosis (n=12) and day 14 post-induction therapy (n=12) with daunorubicin and cytarabine (7+3) were performed. Based on post-treatment frequency of blasts in the AML bone marrow aspirate, patients were classified as responders (<5% blasts) or non-responders (> 5% blasts). Three of 6 responders (3 men, 3 women, average age 59 yrs) had normal karyotype, and three of 6 non-responders (1 man, 5 women, average age 52.6 yrs), had normal karyotype. RNA was isolated from the core bone marrow biopsies and subjected to Clariom D Human Affymetrix arrays. Transcriptomics data were analyzed using Affymetrix Transcriptome Analysis Console with LIMMA R package and Gene Set Enrichment Analysis (GSEA). H&E stained bone marrow biopsy slides were subjected to blinded histopathological assessment. Results: Transcriptomic data analysis of responder vs. non-responder samples at diagnosis indicated significant loss of transcripts associated with heme metabolism (HBB, HBD, GYPE, CA1) suggesting decrease in frequency of erythroid progenitors (Fig.A). Trends of decreased frequency of erythroid progenitors were noted in both bone marrow biopsies and aspirates of diagnostic non-responder samples (Fig.B). Decreased frequency of lymphoid cells was also noted (Fig.B). Interestingly, while post-treatment we noted a relative increase in frequencies of lymphoid cells in both responder and non-responder samples, the increase was more prominent in responders (Fig.B). Trilineage hematopoiesis appeared affected more in diagnostic and post-treatment responder samples. Transcriptome analyses of diagnostic vs. post-treatment responder samples indicated significant increase in transcripts associated with activity within endosteal niche (SPARC, SPP1, DCN, VCAN, BGN) and significant loss of transcripts associated with DNA replication (TOP2, HELLS, E2F8) (Fig.C), the latter was consistent with treatment-related loss of cellularity. Only modest increase in SPARC, SPP1 or BGN levels and no significant decrease in DNA-replication associated transcripts were noted in non-responder post-treatment samples (Fig.1D). These data indicate greater loss of AML cells and greater activity within the endosteal niche in responder in comparison to non-responder samples. Finally, analyses performed on post-treatment responder vs. non-responder samples showed significant decrease in SPARC, SPP1, DCN, VCAN, BGN in non-responder post-treatment samples (Fig.E, F). Endosteal niche in histopathologic evaluation at diagnosis was generally unremarkable in both responder and non-responder samples with only rare osteoblasts present. In contrast, post-treatment, we found an elevated number of osteoblasts in responders in comparison to non-responder samples (Fig.G, H). Conclusions: Transcriptomic and histopathologic analyses of AML bone marrow biopsies procured at diagnosis and post-treatment from responder or non-responders indicate inverse correlation between the activity of endosteal niche and levels of transcripts involved in osteoblast maturation and homeostasis. Significant suppression of mesenchymal/osteoblast component of the niche is observed in non-responder samples. To our knowledge this is a first report showing the correlation between levels of osteopontin (SPP1), osteonectin (SPARC) and biglycan (BGN) and response to chemotherapy directly in the AML core bone marrow biopsies. Our data suggest that osteo-stimulatory factors could be used to achieve better therapeutic outcomes in AML. Disclosures No relevant conflicts of interest to declare.

The Role of Bone Stem Cell Niches in Bone Metastasis

In post-natal life, stem cells contribute to the preservation of many tissues. In adults, stem cells remain localized, in particular, specialized microanatomical areas named as niches, which are crucial in the control of stem cell quiescence and activity through the production of many regulatory signals. Bone physiologically includes both the endosteal niche and the vascular niche, which are involved in the attraction, retention and release of the residing stem cells during the formation of new vessels as well as in the control of the differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. In tumors, cancer cells are able to take control of the niches to support all the stages of the tumorigenesis, transforming them in the so-called pre-metastatic and metastatic niches. Hence, there is emerging importance of the interactions between cancer cells, bone cells and niches in driving metastatic progression. This review article summarizes the literature data concerning the role of bone vascular and endosteal niches in the regulation of bone metastasis, focusing on their cellular and molecular interactions and the potential therapeutic approaches.

Multiple Myeloma as a Bone Disease? The Tissue Disruption-Induced Cell Stochasticity (TiDiS) Theory

The standard model of multiple myeloma (MM) relies on genetic instability in the normal counterparts of MM cells. MM-induced lytic bone lesions are considered as end organ damages. However, bone is a tissue of significance in MM and bone changes could be at the origin/facilitate the emergence of MM. We propose the tissue disruption-induced cell stochasticity (TiDiS) theory for MM oncogenesis that integrates disruption of the microenvironment, differentiation, and genetic alterations. It starts with the observation that the bone marrow endosteal niche controls differentiation. As decrease in cellular stochasticity occurs thanks to cellular interactions in differentiating cells, the initiating role of bone disruption would be in the increase of cellular stochasticity. Thus, in the context of polyclonal activation of B cells, memory B cells and plasmablasts would compete for localizing in endosteal niches with the risk that some cells cannot fully differentiate if they cannot reside in the niche because of a disrupted microenvironment. Therefore, they would remain in an unstable state with residual proliferation, with the risk that subclones may transform into malignant cells. Finally, diagnostic and therapeutic perspectives are provided.

Switching Homes: How Cancer Moves to Bone

Bone metastases (BM) are a very common complication of the most prevalent human cancers. BM are extremely painful and may be life-threatening when associated with hypercalcaemia. BM can lead to kidney failure and cardiac arrhythmias and arrest, but why and how do cancer cells decide to “switch homes” and move to bone? In this review, we will present what answers science has provided so far, with focus on the molecular mechanisms and cellular aspects of well-established findings, such as the concept of “vicious cycle” and “osteolytic” vs. “osteosclerotic” bone metastases; as well as on novel concepts, such as cellular dormancy and extracellular vesicles. At the molecular level, we will focus on hypoxia-associated factors and angiogenesis, the Wnt pathway, parathyroid hormone-related peptide (PTHrP) and chemokines. At the supramolecular/cellular level, we will discuss tumour dormancy, id est the mechanisms through which a small contingent of tumour cells coming from the primary site may be kept dormant in the endosteal niche for many years. Finally, we will present a potential role for the multimolecular mediators known as extracellular vesicles in determining bone-tropism and establishing a premetastatic niche by influencing the bone microenvironment.

Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underlie thrombocytopenia in MYH9-related disease

Megakaryocytes (MKs), the precursor cells for platelets, migrate from the endosteal niche of the bone marrow (BM) toward the vasculature, extending proplatelets into sinusoids, where circulating blood progressively fragments them into platelets. Nonmuscle myosin IIA (NMIIA) heavy chain gene (MYH9) mutations cause macrothrombocytopenia characterized by fewer platelets with larger sizes leading to clotting disorders termed myosin-9–related disorders (MYH9-RDs). MYH9-RD patient MKs have proplatelets with thicker and fewer branches that produce fewer and larger proplatelets, which is phenocopied in mouse Myh9-RD models. Defective proplatelet formation is considered to be the principal mechanism underlying the macrothrombocytopenia phenotype. However, MYH9-RD patient MKs may have other defects, as NMII interactions with actin filaments regulate physiological processes such as chemotaxis, cell migration, and adhesion. How MYH9-RD mutations affect MK migration and adhesion in BM or NMIIA activity and assembly prior to proplatelet production remain unanswered. NMIIA is the only NMII isoform expressed in mature MKs, permitting exploration of these questions without complicating effects of other NMII isoforms. Using mouse models of MYH9-RD (NMIIAR702C+/−GFP+/−, NMIIAD1424N+/−, and NMIIAE1841K+/−) and in vitro assays, we investigated MK distribution in BM, chemotaxis toward stromal-derived factor 1, NMIIA activity, and bipolar filament assembly. Results indicate that different MYH9-RD mutations suppressed MK migration in the BM without compromising bipolar filament formation but led to divergent adhesion phenotypes and NMIIA contractile activities depending on the mutation. We conclude that MYH9-RD mutations impair MK chemotaxis by multiple mechanisms to disrupt migration toward the vasculature, impairing proplatelet release and causing macrothrombocytopenia.

Effects of high glucose conditions on the expansion and differentiation capabilities of mesenchymal stromal cells derived from rat endosteal niche

Background Mesenchymal stromal cells in the endosteal niche lining compact bone (CB-MSCs) represent a heterogeneous population, all of which contribute to bone repair and remodelling. Hyperglycaemia associated with type 2 diabetes mellitus (T2DM) can delay and impair the bone healing process. Therefore, this study investigated the influences of high (25 mM) glucose conditions on CB-MSC populations isolated from male Wistar rats, versus normal (5.5 mM) glucose conditions; in terms of proliferation (population doublings, PDs), senescence characteristics, stem cell marker expression, colony forming efficiencies (CFEs); and osteogenic/adipogenic differentiation, following extended culture in vitro. Results CB-MSCs under both normoglycaemic and hyperglycaemic conditions demonstrated similar morphologies and rapid exponential growth to >300PDs, although high glucose conditions promoted more rapid and persistent proliferation beyond ~50PDs, with few indications of senescence. Limited senescence was confirmed by minimal SA-β-galactosidase staining, low senescence marker (p53, p21waf1, p16INK4a) expression and positive telomere maintenance marker (rTERT, TR) expression. However, telomere lengths varied throughout culture expansion, with hyperglycaemia significantly reducing telomere lengths at PD50 and PD200. Furthermore, CB-MSCs expanded in normal and high glucose conditions remained non-transformed, exhibiting similar MSC (CD73/CD90/CD105), multipotency (CD146) and embryonic (Slug, Snail) markers throughout extended culture, but negligible hematopoietic (CD34/CD45) or pluripotency (Nanog, Oct4) markers. Hyperglycaemia significantly increased CFEs at PD50 and PD100, which decreased at PD200. CB-MSC osteogenic differentiation was also inhibited by hyperglycaemia at PD15, PD100 and PD200, but not at PD50. Hyperglycaemia inhibited CB-MSC adipogenic differentiation to a lesser extent at PD15 and PD50, with reduced adipogenesis overall at PD100 and PD200. Conclusion This study demonstrates the limited negative impact of hyperglycaemia on the proliferative and stem cell characteristics of heterogeneous CB-MSC populations, although minor sub-population(s) appear more susceptible to these conditions leading to impaired osteogenic/adipogenic differentiation capabilities. Such findings potentially highlight the impact of hyperglycaemia on CB-MSC bone repair capabilities in situ.