Macrophages are a vital component of the tumour microenvironment and crucial mediators of tumour progression. In the last decade, significant strides have been made in understanding the crucial ...functional roles played by macrophages in the development of the plasma cell (PC) malignancy, multiple myeloma (MM). Whilst the interaction between MM PC and stromal cells within the bone marrow (BM) microenvironment has been extensively studied, we are only just starting to appreciate the multifaceted roles played by macrophages in disease progression. Accumulating evidence demonstrates that macrophage infiltration is associated with poor overall survival in MM. Indeed, macrophages influence numerous pathways critical for the initiation and progression of MM, including homing of malignant cells to BM, tumour cell growth and survival, drug resistance, angiogenesis and immune suppression. As such, therapeutic strategies aimed at targeting macrophages within the BM niche have promise in the clinical setting. This review will discuss the functions elicited by macrophages throughout different stages of MM and provide a comprehensive evaluation of potential macrophage-targeted therapies.
Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in ...specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse.
Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with ...Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor–induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin−Sca1+KIT+CD41−CD48−CD150+ HSCs segregated into 2 groups (Honeg or Homed), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Honeg HSCs, whereas Homed HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.
Acute myeloid leukaemia (AML), chronic lymphocytic leukaemia (CLL), and multiple myeloma (MM) are age-related haematological malignancies with defined precursor states termed myelodysplastic syndrome ...(MDS), monoclonal B-cell lymphocytosis (MBL), and monoclonal gammopathy of undetermined significance (MGUS), respectively. While the progression from asymptomatic precursor states to malignancy is widely considered to be mediated by the accumulation of genetic mutations in neoplastic haematopoietic cell clones, recent studies suggest that intrinsic genetic changes, alone, may be insufficient to drive the progression to overt malignancy. Notably, studies suggest that extrinsic, microenvironmental changes in the bone marrow (BM) may also promote the transition from these precursor states to active disease. There is now enhanced focus on extrinsic, age-related changes in the BM microenvironment that accompany the development of AML, CLL, and MM. One of the most prominent changes associated with ageing is the accumulation of senescent mesenchymal stromal cells within tissues and organs. In comparison with proliferating cells, senescent cells display an altered profile of secreted factors (secretome), termed the senescence-associated-secretory phenotype (SASP), comprising proteases, inflammatory cytokines, and growth factors that may render the local microenvironment favourable for cancer growth. It is well established that BM mesenchymal stromal cells (BM-MSCs) are key regulators of haematopoietic stem cell maintenance and fate determination. Moreover, there is emerging evidence that BM-MSC senescence may contribute to age-related haematopoietic decline and cancer development. This review explores the association between BM-MSC senescence and the development of haematological malignancies, and the functional role of senescent BM-MSCs in the development of these cancers.
The TWIST family of basic helix‐loop‐helix transcription factors, Twist‐1 and Dermo‐1 are known mediators of mesodermal tissue development and contribute to correct patterning of the skeleton. In ...this study, we demonstrate that freshly purified human bone marrow‐derived mesenchymal stromal/stem cells (MSC) express high levels of Twist‐1 and Dermo‐1 which are downregulated following ex vivo expansion. Enforced expression of Twist‐1 or Dermo‐1 in human MSC cultures increased expression of the MSC marker, STRO‐1, and the early osteogenic transcription factors, Runx2 and Msx2. Conversely, overexpression of Twist‐1 and Dermo‐1 was associated with a decrease in the gene expression of osteoblast‐associated markers, bone morphogenic protein‐2, bone sialoprotein, osteopontin, alkaline phosphatase and osteocalcin. High expressing Twist‐1 or Dermo‐1 MSC lines exhibited an enhanced proliferative potential of approximately 2.5‐fold compared with control MSC populations that were associated with elevated levels of Id‐1 and Id‐2 gene expression. Functional studies demonstrated that high expressing Twist‐1 and Dermo‐1 MSC displayed a decreased capacity for osteo/chondrogenic differentiation and an enhanced capacity to undergo adipogenesis. These findings implicate the TWIST gene family members as potential mediators of MSC self‐renewal and lineage commitment in postnatal skeletal tissues by exerting their effects on genes involved in the early stages of bone development. STEM CELLS 2009;27:2457–2468
Multiple myeloma is an incurable hematologic cancer characterized by the clonal proliferation of malignant plasma cells within the bone marrow. Numerous studies suggest that the myeloma plasma cells ...occupy and alter the stromal tissue of the bone marrow as a means of enhancing their survival and growth. However, the nature and magnitude of the changes to the stromal cell tissue remain to be determined. In this study, we used mesenchymal stromal cell and osteoblast-related cell surface marker expression (STRO-1 and alkaline phosphatase, respectively) and flow cytometry to enumerate mesenchymal stromal cell and osteoblast numbers in bone marrow recovered from myeloma patients at the time of diagnosis. Using this approach, we identified an increase in the number of STRO-1 positive colony forming mesenchymal stromal cells and a concomitant decrease in alkaline phophatase osteoblasts. Notably, this increase in mesenchymal stromal cell numbers correlated closely with plasma cell burden at the time of diagnosis. In addition, in comparison with the osteoblast population, the STRO-1+ mesenchymal stromal cell population was found to express higher levels of plasma cell- and osteoclast-activating factors, including RANKL and IL-6, providing a mechanism by which an increase in mesenchymal stromal cells may promote and aid the progression of myeloma. Importantly, these findings were faithfully replicated in the C57BL/KaLwRij murine model of myeloma, suggesting that this model may present a unique and clinically relevant system in which to identify and therapeutically modulate the bone microenvironment and, in turn, alter the progression of myeloma disease.
Since its discovery more than 25 years ago, the STRO‐1 antibody has played a fundamental role in defining the hierarchical nature of mesenchymal precursor cells (MPC) and their progeny. STRO‐1 ...antibody binding remains a hallmark of immature pluripotent MPC. Despite the significance of STRO‐1 in the MPC field, the identity of the antigen has remained elusive. Using a combination of two‐dimensional gel electrophoresis, coupled with Western blotting and Tandem mass spectroscopy, we have identified the STRO‐1 antigen as heat shock cognate 70 (HSC70;HSPA8). STRO‐1 binds to immune‐precipitated HSC70 and siRNA‐mediated knock down of HSPA8 reduced STRO‐1 binding. STRO‐1 surface binding does not correlate with HSC70 expression and sequestration of cholesterol reduces STRO‐1 surface binding, suggesting that the plasma membrane lipid composition may be an important determinant in the presentation of HSC70 on the cell surface. HSC70 is present on the surface of STRO‐1+ but not STRO‐1− cell lines as assessed by cell surface biotinylation and recombinant HSC70 blocks STRO‐1 binding to the cell surface. The STRO‐1 epitope on HSC70 was mapped to the ATPase domain using a series of deletion mutants in combination with peptide arrays. Deletion of the first four amino acids of the consensus epitope negated STRO‐1 binding. Notably, in addition to HSC70, STRO‐1 cross‐reacts with heat shock protein 70 (HSP70), however all the clonogenic cell activity is restricted to the STRO‐1BRIGHT/HSP70− fraction. These results provide important insight into the properties that define multipotent MPC and provide the impetus to explore the role of cell surface HSC70 in MPC biology. Stem Cells 2017;35:940–951
Cell surface HSC70, as detected by STRO‐1 antibody binding, is localized to lamellipodia like cell protrusions in UE7T‐13 cells, an immortalized human bone marrow derived mesenchymal stromal cell line.
The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in ...clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
•Dormant multiple myeloma cells express a unique transcriptome signature.•This unique transcriptome signature controls dormancy, predicts survival, and identifies new treatment targets.
Display omitted
Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have ...isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.
Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse ...bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.
Display omitted
•Genuine macrophages are absent from hematopoietic tissue single-cell preparations•Fragmented remnants of macrophages containing cytoplasm adhere to other cells•Macrophage remnants convolute hematopoietic tissue flow cytometry analysis•Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data
Millard et al. demonstrate intact macrophages are absent in hematopoietic tissue single-cell suspensions as they are disrupted during preparation. Macrophage remnants containing membrane and intracellular contents remain attached to other cells within the suspensions. This results in misassignment of macrophage identity and misattribution of macrophage-expressed genes.
PDF
