The bone marrow niche: habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites

Abstract

In post-fetal life, hematopoiesis occurs in unique microenvironments or ‘niches’ in the marrow. Niches facilitate the maintenance of hematopoietic stem cells (HSCs) as unipotent, while supporting lineage commitment of the expanding blood populations. As the physical locale that regulates HSC function, the niche function is vitally important to the survival of the organism. This places considerable selective pressure on HSCs, as only those that are able to engage the niche in the appropriate context are likely to be maintained as stem cells. Since niches are central regulators of stem cell function, it is not surprising that molecular parasites like neoplasms are likely to seek out opportunities to harvest resources from the niche environment. As such, the niche may unwittingly participate in tumorigenesis as a leukemic or neoplastic niche. The niche may also promote metastasis or chemo-resistance of hematogenous neoplasms or solid tumors. This review focuses on what is known about the physical structures of the niche, how the niche participates in hematopoiesis and neoplastic growth and what molecules are involved. Further understanding of the interactions between stem cells and the niche may be useful for developing therapeutic strategies.

Introduction

For species to survive and reproduce to maintain their populations, they must harvest resources from and be in tune with the environment that they find themselves in. Many combinations of environmental factors are necessary to prosper in the physical environment, to obtain energy and nutrients, to avoid predation and to ultimately pass on genetic material to progeny. These requirements are abstractly termed the ‘ecological niche’. Historically, Grinnell proposed the ‘niche’ term as a collection of environment conditions where a species lives.¹ Hutchinson made the ecological niche concept popular by suggesting that the niche could be modeled as an imaginary space with many dimensions.² In mammals, Schofield proposed the niche hypothesis to describe the microenvironment that supports hematopoietic stem cells (HSCs).³

In the marrow, bone marrow stromal cells derived from mesenchymal stem cells (MSCs) are believed to provide the basis for the physical structures of the niche. As such, bone marrow stromal cells are thought to regulate self-renewal, proliferation and differentiation of the HSCs through production of cytokines and intracellular signals that are initiated by cell-to-cell adhesive interaction.⁴ Bone marrow stromal cells are composed of cells of mesenchymal origin known to include osteoblasts, endothelial cells, fibroblasts and adipocytes.⁵ As early as the 1970s, studies examining the interaction between bone marrow stromal cells and HSCs had already suggested a unique and critical role for events localized at endosteal surfaces, and by inference osteoblasts, as having a central role in hematopoiesis.^6,7 Only recently have studies demonstrated that osteoblasts comprise a crucial component of the HSC niche (‘endosteal niche’),^8–11 while cells of other lineages including endothelial cells (for example, ‘vascular niche’) are also likely to participate in niche functions.^12–15 At the same time, there is a growing appreciation from studies examining the physical locale of where MSCs reside in marrow that they too may localize to similar niches in marrow¹⁶ (Figure 1a). As such, it is very likely that HSCs and MSCs are intimately associated with one another. Moreover, it is likely that the co-coordinated activities of the niche (be it endosteal or endothelial) and all the stem cells collectively are responsible for integrating the delicate balance between hematopoiesis and tissue turnover.

Figure 1.

(a) A model of bone marrow niches. In the marrow, osteoblasts and endothelial cells constitute the major cellular components contributing to the endosteal and vascular niches that serve as the microenvironment for maintaining hematopoietic stem cells (HSCs; ‘HSC niche’). Osteoblasts and endothelial cells are derived from mesenchymal stem cells (MSCs) and hemangioblasts, respectively. Recent data suggest that MSCs themselves may reside in niches that are in close proximity to the HSC niche. In addition, there is growing evidence that HSCs and MSC co-regulate activities of each other. In the model presented, overlap of HSC, MSC, vascular and endosteal niche function occurs and is required for the coordinated function of the marrow. (b) A model of the neoplastic niche. Increasing evidence suggests that disseminated tumor stem cells reside in niches that facilitate the metastasis and survival of tumors in distant tissues. Much like HSCs and MSCs, the residence of metastatic cells in the niche provides signals that regulate dormancy and escape from chemotherapy and radiotherapy. As such, the residence of metastatic cells in the niche constitutes a molecular parasite of the normal host regulatory functions that exist to supply a constant flux of HSC and MSC progeny throughout the lifetime of the individual.

Many solid tissue neoplasms also have a predisposition to metastasize to the marrow, including breast and prostate cancers (PCa).¹⁷ Multiple factors are involved in the metastatic spread of neoplasms to the marrow. Potentially these tumor cells have the ability to respond to niche localization signals that promote extravasation from primary sites as well as the preferential adhesion to bone marrow endothelium. In addition, the niche may add to or participate in the selection of malignant clones that facilitate the adaptation of cancer cells to grow within bone marrow microenvironments.^17,18 On the basis of our studies relating to the HSCs homing, we have recently reported the significant role of the chemokine CXC chemokine ligand 12 (CXCL12 or SDF-1)/its major receptor CXC chemokine receptor 4 (CXCR4) axis on the bone metastasis of Pca.^19–23 Although the mechanisms of bone metastasis are still unclear, the CXCL12/CXCR4 chemokine axis is likely to play a significant role in regulating the bone metastasis of solid neoplasms. Thus, several lines of evidence demonstrate that bone marrow stromal cells are also able to promote the growth, survival and drug resistance of hematogenous neoplasms.^24–26 These findings suggest that the molecular mechanisms used by the niche to maintain quiescence of stem cells may be parasitized to facilitate neoplastic growth.²⁷

In this review, we focus on the role of bone marrow stromal cells, with a particular focus on osteoblasts, in the HSC niche. We also explore and develop the hypothesis that neoplasms function as molecular parasites on the niche (Figure 1b). By taking advantage of the normal machinery used by the niche to maintain stem cells as stem cells, we believe that tumors are able to survive in the marrow for long periods of time. Therefore, the development of niche-directed therapies would add to the armamentarium of therapies for leukemia and bone metastasis of solid neoplasms.

Hematopoietic stem cells niche

In adults, marrow-derived HSCs are the principle cells of origin of all mature hematopoietic cell phenotypes. Hematopoiesis is considered as a pyramidal/hierarchical process with the cells’ of greatest maturation potential or primitiveness sitting at the top of the hierarchy, and cells that have undergone terminal differentiation at the bottom. Terminally differentiated blood cells are generally classified into one of two major lineages: those derived from myeloid lineages and those derived from lymphoid progenitors. Myeloid cells include red blood cells, platelets and cells responsible for cellular immunity, such as macrophages and granulocytes. Cells derived from lymphoid progenitors are major participants in coordinating humeral and cellular immunity.

Experimental data suggest that HSCs differentiate into hematopoietic progenitor cells that are capable of exponential proliferation as well as continuing the process of differentiation. Alternatively, HSCs are capable of self-replicate activity, which may give rise to one or two identical daughter cells. As a result, HSC activity must be tightly regulated so as to meet physiologic demands, but also to protect HSCs from oncogenic, physical and chemical damage. The site or physical locale that regulates self-renewal, proliferation and differentiation of HSCs has been termed the ‘HSC niche’ (Figure 2). The niche is likely comprised of many different niche constituents including osteoblasts, endothelial cells, fibroblasts and adipocytes. Other cells known to participate in the generation of the niche or its regulation are osteoclasts and the HSCs and progenitor cells themselves—which is only now becoming appreciated.²⁸

Figure 2.

A model of the hematopoietic stem cell (HSC) niche in long bones. (a and b) Organization of long bones and location of the HSC niches. Recent studies demonstrate that endosteal osteoblasts and their precursors play a critical role the stem cell ‘niche’ (c). In addition, endothelial cells are likely to contribute to niche function (c). Central to these hypotheses are the demonstration of osteoblast (OB)- or endothelial cell-expressed regulatory components that influence stem cell function are likely to include cell-to-cell receptors, and soluble and cell-surface associated cytokines and growth factors. (d) Egress into and out of the marrow by HSCs facilitate transit into and out of the vascular niche, which is permissive for proliferation and differentiation. The endosteal niche facilitates HSC maintenance and quiescence. Reciprocal interactions between stem cells and their niches are likely to play key roles in the establishment and maintenance of the stem cell niche in the bone marrow (not shown). (e) Factors produced by osteoblasts that influence HSC. Stem cell fate is influenced by specialized microenvironments that remain poorly defined. Osteoblasts produce soluble hematopoietic-supportive secreted and cell-associated factors that work in concert so that HSCs derive regulatory information from bone, accounting for the localization of hematopoiesis in bone marrow (homing/localization receptors e.g. Annexin II, VCAM-1, CXC chemokine receptor 4 (CXCR4)/CXC chemokine ligand 12 (CXCL12)). Quiescence factors (e.g. bone morphogenic factors, fibroblast growth factors, Flt-3 ligand, Tie2/Ang-1, granulocyte and granulocyte-macrophage colony-stimulating factors, hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), osteopontin and high levels of extracellular calcium) and cell-associated factors are known to include but are limited to osteopontin, granulocyte and granulocyte-macrophage colony-stimulating factors and transforming growth factors (TGFs). Soluble factors known to influence HSC function include parathyroid hormone and erythropoietin (Epo).

Osteoblasts

As late as 1992, virtually nothing was known regarding the role of osteoblasts in hematopoiesis. What was published was focused on what cytokines/growth factors were produced by osteoblast, the role that osteoblasts play in regulating osteoclastic activities or on the role that immortal cell lines play in hematopoiesis.²⁹ Since this time, our group has demonstrated that osteoblasts support hematopoietic progenitor and long-term culture-initiating cell (LTC-IC) activities in a human system.^30–32 Moreover, the proximity of CD34-positive bone marrow cells to osteoblasts can induce the synthesis of several cytokines, including interleukin (IL)-6,³³ leukemia inhibitory factor (LIF),³³ transforming growth factor-β1 (TGF-β1),³¹ macrophage inhibitory protein-1α,³⁴ hepatocyte growth factor (HGF),³² CXCL12,³⁵ and IL-7.³⁵ However, progenitor cells and LTC-IC survival on osteoblasts requires direct cell-to-cell contact, despite the elaboration of soluble cytokines that are necessary, but clearly not sufficient for maintenance of hematopoietic cells on osteoblasts.³⁰ HSCs also bind tightly to osteoblasts in vitro, and that these interactions were sensitive to trypsin, chelators and glycosylation inhibitors.³⁶ These data suggest that direct cell-to-cell contact of HSCs on osteoblasts is critical to ensure HSCs survival (Figure 2). Although osteoblasts express many cell adhesion molecules that may be used during HSCs–osteoblasts adhesion, the molecular mechanisms resulting the interactions between HSCs and osteoblasts are not well defined.²⁹ In recent work, we have demonstrated that the adhesion between HSCs and osteoblasts is mediated by cell-to-cell rather than cell-to-matrix.^5,36 Moreover, two separate types of adhesive relationships seem to exist.

One group of cell adhesion molecules regulates the immediate binding of HSCs homing to the endosteal niche. Rapidly processed N-linked glycosylated proteins constitutively expressed on HSCs, as detected using selective pretreatment with glycosylation inhibitors, are most likely involved.³⁶ CXCL12/CXCR4 interactions are also thought to regulate localization of HSCs to endosteal niche,^37,38 and play a major role in granulocyte colony-stimulating factor (G-CSF) induced HSCs mobilization.³⁹ CXCL12 is a critical component of the niche that appears to be a pivotal point of regulation of cell entry and egress from the marrow. The interactions between CXCL12 and its major receptor CXCR4 are thought to play an important role in normal hematopoietic development.^17,40 Animal studies have shown significant defects in the marrow function in the absence of CXCL12 or CXCR4.^41–44 In addition, blockade of CXCR4 prevents HSC homing to the marrow.⁴⁵ CXCL12 is constitutively produced by many bone marrow stromal cells including endothelial cells and fibroblasts, although osteoblasts are likely to be the major a source of the protein under basal conditions.^5,37,38 Yet, other cells may in fact be capable of expressing CXCL12 and presenting it to HSCs including the recently described CXCL12-abundant reticular cells.⁴⁶ Our recent data also demonstrate that annexin II plays an important role in the localization of HSCs to the endosteal niche⁴⁷ (Figure 2). Such that blocking annexin II with antibody or competing peptide prevents engraftment and survival in lethally irradiated animals.⁴⁷

Another group of adhesion molecules probably provides survival signals or generates activation of a quiescent state in HSCs after they are localized to the endosteal niche. These are known to include ostepontin (OPN),^48,49 very late antigen-4 (VLA-4)/fibronectin (FN) or vascular cell adhesion molecule-1 (VCAM-1),⁵⁰ leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1),⁵⁰ and N-cadherin.^9,11 (Figure 2)

Although HSCs and osteoblasts are closely associated with each other in the bone marrow, and osteoblasts modulate normal hematopoiesis,⁵ the details of the post-receptor engagement events that regulates HSC quiescence remains largely unexplored. One pathway that has garnered considerable attention is the N-cadherin and β-catenin pathway, are believed to be in part responsible for interaction between osteoblasts and long-term HSCs,⁹ although recent reports suggest that HSCs themselves may not express N-cadherin.⁵¹ Likewise, activation of osteoblasts through the parathyroid hormone receptor (PTHr) has been shown to expand HSCs number.¹⁰ Here, osteoblasts are thought to regulate HSCs number through Notch1/Jagged1 pathway signaling.¹⁰ Likewise, the tight adhesion between HSCs and osteoblasts induces Tie2/angiopietin 1(Ang-1) signaling is likely to result in the maintenance of HSCs in a quiescent state in the bone marrow niche.¹¹ Along these lines, most recently we demonstrated that osteoblasts directly support B lymphopoiesis through VLA-4/VCAM-1 interaction³⁵ as a function of osteoblasts to regulate the differentiation of HSCs. These findings strongly suggest a relationship between osteoblasts and hematopoietic cells, but the dimension of this interaction has, yet, to be completely defined.

Endothelial cells

Recent work by several groups suggest that not only do bone marrow endothelial serve to partition the vascular and extra-vascular marrow spaces, but they also regulate HSCs by functioning in a niche capacity. The vast majority of the studies support that an endothelial marrow niche does so on account of the localization of HSCs by immunohistochemical analysis. The most eloquent being the use of simple combinations of markers, which are termed ‘SLAM’ family receptors, including CD150, CD244 and CD48, which identified the majority of HSCs in close physical association with endothelial cells in vivo.¹² The presence of a bone marrow endothelial cell, or ‘vascular niches’ for HSCs makes inherent sense in that in order to rapidly mobilize large quantities of stem cells into the periphery they would need to be in position and ready to respond to systemic challenges. However, a substantial body of literature has demonstrated that transplanted HSCs localize rapidly to the endosteum,^48,52 and marrow dissection studies provide similar information.^53,54 Yet, whether there is a circulation or communication between the two niches (vascular and endosteal), has been difficult to identify directly.¹⁵

What has been dissected from the system is that CXCR4 expressed on the bone marrow endothelial cells plays an important role in the homing of human CD34-positive hematopoietic progenitors to the bone marrow by translocation of circulating CXCL12 into the bone marrow.¹³ Bone marrow endothelial cells also induce HSCs proliferation and expansion by secreting the growth factors such as insulin-like growth factor binding protein-3 (IGFBP-3).¹⁴ Thus, endothelial cells might regulate HSC function and could contribute to the creation of HSC niches as they do in the nervous system.¹²

Like their role in supporting HSCs, bone marrow endothelial cells play a critical role in thrombopoiesis.⁵⁵ CXCL12 and fibroblast growth factor-4 (FGF-4) induced the localization and adhesion of megakaryocytes to the bone marrow endothelial cells.⁵⁵ The direct contact between megakaryocyte and bone marrow endothelial cells promotes survival, maturation and release of platelets through VE-cadherin and/or VLA-4/VCAM-1 axis.⁵⁵ Similarly, the VLA-4/VCAM-1 axis is likely to play a critical role in HSC trafficking.

Although it is still controversial, several reports suggest that HSCs in different niches are in different states of quiescence. One attractive model for HSCs maintenance and expansion is that osteoblasts mainly maintain the quiescent state of HSCs, while HSCs differentiate and proliferate on endothelial cells⁵⁶ (Figure 2). If true, HSCs in an endosteal niche may be more deeply quiescent than in vascular niches. Such that in response to stress as in infection, allergy or bleeding, HSCs are able to enter into replicative states in an vascular niche or migrate to an vascular niche, and are capable of entering the circulation. This process, in reverse would provide a mechanism for induction of quiescence in endosteal sites or niches. Of course this hypothesis is highly speculative and an area of intense investigation and debate.

Mesenchymal stem cells

MSCs have the potential to differentiate into specialized cells of mesenchymal origin, including adipocytes, chondrocytes, myoblasts and osteoblasts.^57–63 However, MSCs are present at low frequency in bone marrow and in the circulation.⁶⁴ Despite the lack of cellular and molecular tools and in vivo assays that permit the rigorous identification of stem cells from mesenchymal tissues, it is known that a cell population with MSC-like characteristics can be isolated based on its ability to adhere to plastic and may be partially purified by separation techniques.^65–68 MSCs can be expanded to great numbers by in vitro culture, however, such a step is time consuming, expensive and risks cell contamination. Most importantly, as these cells gain or lose their differentiation potential during long-term culture, they likely acquire properties that do not appear under physiologic, in vivo circumstances. Therefore, studies have demonstrated that pure MSCs are quite rare. Furthermore, limiting our efforts to in vitro studies aimed at expanding MSCs without adequate in vivo assays hinder the rigorous identification of stem cells from mesenchymal tissues. In parallel, our in vivo work by referring to other work⁶⁹ has demonstrated that a 5-fluorouracil (5-FU) resistant population of mesenchymal cells is capable of multi-lineage differentiation. Likewise, it has recently been demonstrated that a CD146-positive cells in human marrow is able to perform a similar function in vivo.⁷⁰

It is known that the endosteal/subendosteal microenvironment includes bone lining and adjacent reticular cells that support HSC self-renewal, proliferation and differentiation. Recently, a technique was developed to fractionate the marrow using differential digestion.¹⁶ The two distinct stromal-cell populations containing the fraction of subendosteal reticulocytes and osteoblasts were isolated from periosteum-free fragments of murine femurs by a two-step collagenase-digestion procedure. Both populations produce similar extracellular matrix (collagen I, laminin, FN and decorin), except for collagen IV, which is low in a fraction of subendosteal osteoblasts. These populations are distinctly different in their osteoblastic characteristics in vitro compared to marrow that is flushed (whole bone marrow). In conjunction with preconditioning mice with 5-FU, we subsequently demonstrated that the fraction of subendosteal reticulocytes population had a greater osteogenic potential than the other marrow fractions in vitro and in vivo animal model⁷¹ (in preparation).

For identification of HSCs, the answer has been to eliminate the tissue through lethal levels of radiation. For MSCs or their immediate progeny, this approach is not feasible due to the lack of an assay comparable to the competitive reconstitution assay. However, recent work described a method of multiparameter cell sorting that yielded a population of cells that resembled very primitive, in fact, embryonic-like cells when cultured.⁷² These cells develop into neurospheres and their potential for tissue/organ regeneration appears to be unlimited.⁷² It has also been identified as a population of CXCR4-positive/lineage-negative/CD45-negative cells that express SSEA, Oct-4 and Nanog in adult bone marrow.⁷³ These cells are extremely small and display several features typical for primary embryonic stem cells. These cells include (1) a large nuclei surrounded by a narrow rim of cytoplasm; (2) open-type chromatin (euchromatin) and (3) high telomerase activity.⁷³ In in vitro cultures, these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in the bone marrow from young (approximately 1-month old) mice and decreases with age. It is also observed that significantly fewer cells are found in the short-living DBA/2J mice as compared to long-living B6 animals. These cells strongly respond in vitro to CXCL12, HGF/scatter factor and LIF.⁷² They also express CXCR4, c-met and LIF receptor.⁷² The authors named these cells very small embryonic-like (VSEL) stem cells, and hypothesized that they are direct descendants of the germ lineage. These combinations of markers go along with the concept that these cells may in fact be embryonic stem cells, epiblast stem cells and primordial germ cells.^72–77 These cells could also give rise to MSCs down the road. Thus, VSEL cells could reside on the top of the MSC hierarchy in bone marrow. Moreover, the presence of these stem cells in adult tissues including bone marrow, epidermis, bronchial epithelium, myocardium, pancreas and testes supports the concept that adult tissues contain some population of pluripotent stem cells that are deposited in embryogenesis during early gastrulation. In fact, the cells deposited in the marrow early during ontogenesis and can be mobilized from bone marrow and circulate in peripheral blood during tissue/organ injury in an attempt to regenerate damaged organs.

Further studies have demonstrated that VESL cells, when isolated freshly from bone marrow, neither grow hematopoietic colonies nor radioprotect lethally irradiated recipients. However, when VSEL cells are co-cultured with C2C12 murine sarcoma-supportive feeder layers, the sphere formal cells, which are composed of immature cells with large nuclei containing euchromatin and express other VSEL cell-like properties, can be subcultured⁷⁸ After replating these cells, new embryonic-like bodies were formed. If plated into cultures promoting tissue differentiation, VSEL cells show pluripotency and expand into cells from all three germ-cell layers. In this case, however, CD45-negative VSEL, if cultured/passaged in methylocellulose cultures supplemented with hematopoietic growth factors (c-kit ligand, IL-3, erythropoietin (Epo) and granulocyte-macrophage colony-stimulating factor (GM-CSF)), give rise to colonies composed of myeloid (CD45⁻positive/Gr-1-positive) and erythroid (Ter119-positive) hematopoietic cells. The hematopoietic differentiation of VSEL cells was accompanied by upregulation of mRNA for several genes regulating hematopoiesis (for example, PU-1, c-myb, LMO2 and Ikaros).

From the standpoint of the niche, VSEL cells are highly mobile and respond to a CXCL12 gradient and adhere to bone marrow-derived fibroblasts. Time-lapse studies reveal that the cells attach to and migrate beneath and/or undergo emperipolesis in bone marrow stromal cells. Interaction of VSEL cells with bone marrow stromal cells is inhibited after preincubation of the cells with CXCR4 antagonists. Since bone marrow stromal cells secrete CXCL12 and other chemoattractants, stromal cells may create a homing environment for VSEL cells. Thus, the interaction of VSEL cells with MSCs that are isolated from bone marrow stromal cells may, in fact, have been ‘contaminated’ by VSEL cells including the so-called multipotential adult progenitor cells, unrestricted somatic stem cells or marrow-isolated adult multilineage inducible cell cultures.⁷⁹ On the basis of these observations and observations that bone marrow stromal cells support HSC growth and differentiation, it is clear that ‘MSCs’ reside in bone marrow in a locale that is similar to HSCs (Figure 1a).

In a parallel line of work, it has been demonstrated that co-transplantation of enriched MSCs with HSCs improves engraftment and reduces graft failure (or graft-versus-host disease).^80–82 Likewise, co-culture of HSCs with mixed MSCs facilitates the expansion of HSCs ex vivo to a limited degree.^83–85 Consequently, MSCs are likely to be an essential component of HSC niche, as well as osteoblasts and endothelial cells. Part of the mechanism may be that MSCs provide support during hematopoiesis through the abundant production of hematopoietic-supportive molecules such as fibronectin, osteopontin, CXCL12, Ang-1, thrombospondin and others.⁸⁶ MSCs are also rich sources of other hematopoietic active cytokines including but not limited to IL-6, −7, −8, −11, −12, −14, −15, LIF, macrophage (M)-CSF, fms-like tyrosine kinase-3-ligand (Flt3-L) and stem cell factor.⁸⁷ Yet, the production of these molecules by MSCs has not been clearly identified.

Besides expanding HSCs, MSCs can support T-cell ^86,88,89 and B-cell^88,90 survival in a quiescent condition by preventing apoptosis. In addition, MSCs inhibits dendritic cell differentiation^91,92 by inducing cell cycle arrest. By inference with the activities of MSCs then, the main roles of MSCs in hematopoiesis are thought to regulate the immune system. Although the role of the niche as an entity that regulates immunity is relatively unexplored, the concept that MSCs may regulate engraftment and event metastasis is clearly an area in need of further investigation.⁹³

Molecular parasites of the niche

It was recently proposed that most cancers contain a rare population of functionally distinct cancer stem cell (CSC). One of the first neoplasms in which a stem cell was identified was acute myeloid leukemia (AML).⁹⁴ In this disease, the frequency of the leukemic stem cell (LSC) is approximately one per million AML blasts where a CD34-positive/-negative cell fraction representing 0.1–1% of the neoplasm cells possessed all the leukemia-initiating activity in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation model.⁹⁴ A mammary CSC has recently been isolated from primary mammary carcinomas using four cell-surface markers (CD44, CD24, a mammary cancer marker and epithelial-specific antigen).⁹⁵ The neoplasm-initiating capacity of the cells was also verified in an in vivo NOD/SCID engraftment assay. The mammary CSCs represented approximately 2% of the unfractionated bulk neoplasm cells.

As was described previously, stem cells generally reside in a microenvironmental niche, where niche regulates proliferation, differentiation and self-renewal of the stem cells. However, an open question is where in marrow the CSC niche is and how it does function. An equally important question to discern whether or not, the ‘normal’ niche is one and the same with those targeted by metastatic cells. If so, does the function of the niche itself contribute to metastasis? Intuition would suggest engagement of the niche by similar adhesive mechanism used by HSC to localize to the niche would facilitate neoplastic dissemination.

We have approached the issue from the perspective of PCa, because PCa is a common cancer and the second leading cause of cancer deaths in American men.^19,20,96,97 The high-mortality rate is principally attributable to the spread of malignant cells to many tissues including bone.¹⁹ Although the most common metastatic site of PCa is the bone, there have not been many advances in the therapeutic arena to prevent or diminish these lesions. Bone provides chemotactic factors, adhesion factors and growth factors that allow the PCa to target and proliferate in the bone.⁹⁶ CXCL12 is thought to activate proliferation of PCa cells.^20,21 On the other hand, neoplasm-derived factors have direct effects on osteoblasts differentiation and proliferation, the response of bone to neoplastic invasion and bone homeostasis. In a reciprocal relationship, PCa is believed to supply osteoblastic growth factors, such as vascular endothelial growth factor (VEGF)⁹⁸ and osteolytic factors that modulate bone remodeling.⁹⁶ At the same time, while osteoblastic activity is clearly the predominant feature of late stage of PCa metastasis, there is a significant component of osteoclastic activity in PCa. PCa-induced osteolysis may be due to both inhibitory effects on osteoblasts and stimulatory effects on osteoclasts by enhanced OPN secretion by osteoblasts.⁹⁹ We have also reported that the CXCL12/CXCR4 chemokine axis plays a significant role in the bone metastasis of PCa,^19–23 by activating αvβ3 integrins²³ and CD164.²² CD164 also may play an important role in localizing tumors not only to sites where there are high levels of CXCL12 expression, but also to specific tissue locales.²² CXCR4 expression is correlated with neoplastic grade,²⁰ and that CXCL12 signaling through CXCR4 triggers the adhesion of PCa cells to bone marrow endothelial cells.¹⁹ Recently we also found that the CXCL12/CXCR4 chemokine axis plays a significant role in secreting proangiogenic signals, such as IL-6, −8, tissue inhibitors of metalloproteinase (TIMP)-2 and VEGF, through MEK/extracellular signal-regulated kinase (ERK) or PI-3K/AKT pathway and in regulating expression of angiostain levels by using short interfering RNA knockdown technology for CXCR4.⁹⁷ Moreover osteoblasts are known to regulate PCa growth and survival. As such, PCa and osteoblasts regulate activities of each other, much like the relationship between HSCs and the endosteal niche.

An other open question is what does engagement of the niche mean specifically for tumors? One would expect that activation of the programs that are used by the niche to regulate quiescence in HSCs and possibly MSCs would result in the induction of dormancy programs in neoplasms. LSCs share many of the properties of HSCs including aspects pertaining to self-renewal, differentiation and lineage commitment pathways. As was detailed earlier, several members of the bone marrow stromal-cell family contribute either physically or functionally to the HSC niche. Likewise, bone marrow stromal cells control the fate of LSCs, including quiescence, resistance to apoptotic stimuli and resistance to drugs. These pathways are regulated most efficiently when LSCs are in near proximity to bone marrow stromal cells or in direct contact.¹⁰⁰ For example, direct cell-to-cell contact inhibits leukemic cells from cell growth and activates cell survival signaling pathways leading to the protection of LSCs from chemotherapy-induced apoptotic cell death.¹⁰¹

Although the molecular mechanisms which regulate LSC self-renewal remain unclear, a recent study demonstrated that asparagine, which is secreted by bone marrow stromal cells in large amounts, protected acute lymphoblastic leukemia (ALL) cells from asparaginase treatment in co-culture systems.¹⁰² Some adhesion molecules such as VCAM-1, FN, hyaluronic acid and VE-cadherin were demonstrated to have prosurvival activity in leukemia.^24,103–106 Along these lines, direct cell-to-cell contact between leukemia and the niche may not only contribute to cell adhesion, but mediate resistance to chemotheraputic agents. For example, adhesion of B-lineage ALL cells to bone marrow stromal cells provides protection from cytarabine- and etposide-induced apoptosis through the VLA-4/VCAM-1 interactions.¹⁰¹ VLA-4/FN interactions have also been shown to regulate drug resistance in AML by altering the PI-3K/AKT signaling pathways to compensate for interruption of VLA-4 signaling in the presence of function-blocking antibodies.¹⁰⁷ Likewise, CD44 is thought to play a key function in AML such that antibody to the molecule inhibits AML homing to the marrow.¹⁰⁸ Growth arrest following adhesive contacts between leukemic cells and the stromal niche appears to be critical juncture in protecting leukemic cells from drugs that induce apoptosis. Although microenvironment for leukemia is still unclear, a recent study has demonstrated that osteoblasts are capable of serving in a niche capacity for leukemic cells.²⁷ The bone marrow is thought to be not only primary site of leukemia, but also one of the sites for leukemic relapse. CXCL12/CXCR4 interactions provide ALL cells a pathway for homing to endothelial cells in the bone marrow, and subsequently an opportunity to engage the niche.¹⁰⁹ Conversely, leukemic cells actively recruit bone marrow endothelium to establish vascular networks suitable for continued growth via a CXCL12/CXCR4 pathway.^40,110 What are the molecules involved, and how osteoblasts regulate the leukemic niche is far from understood. As mentioned in the context of HSCs, osteoblasts produce a vast number of cell-associated and -secreted proteins that may induce growth arrest of leukemic cells.⁵ Yet, none are likely to be sufficient alone. Clearly, a better understanding of the molecular mechanisms regulating the interactions between the niche and its constituents and leukemic cells will lead to new possibilities for therapeutic interventions in patients with minimal residual disease.

In view of these observations, it seems reasonable to surmise that many of the molecular mechanisms that enable self-renewal of HSCs may be shared with LSCs and CSCs (Figure 1b). One possibility is that the number of niches is limited, and therefore as tumor burden increases, the capacity for regulation is eventually overwhelmed. Under such a scenario, it is appealing to speculate that establishment of niche regulation, or expansion of the niches may be attractive therapeutic targets.

Summary

The bone marrow stromal cells provide survival signals to HSCs, LSCs and CSCs probably through similar pathways. Yet, although a number of similarities exist between normal HSCs, LSCs and CSCs in terms of molecular mechanisms that regulate self-renewal and interactions with the bone marrow microenvironment, clearly differences exist as well^111,112 (Table 1). One such mechanism that was recently proposed is differences in microRNAs, small nonprotein-coding RNAs, believed to regulate the self-renewal of stem cells. The loss of microRNAs in aging of stem cells may promote tumorigenesis.¹¹³ The role of bone marrow stromal cells in protecting leukemic cells from chemotherapy through cell-to-cell adhesion has been widely recognized. One possibility is that LSCs have much more active migration machinery when compared with HSCs that allow them to escape growth inhibition or quiescence promoting signals induced by osteoblasts and other stromal cells in the niche. Thus, drugs that target adhesion molecules combining with current chemotherapy hold certain promise as the next step of therapeutic regimens to leukemia. Similar to hematopoiesis and leukemia, bone marrow stromal cells might involve in creating CSCs through direct cell-to-cell contact as a metastatic site. Although the present chemotherapies destroy the bulk of a tumor, they do not target specifically CSCs that may be slowly cycling especially if they are engaged in the niche (Figure 3). Therefore, a next step of cancer chemotherapy will most likely be to target CSCs. On the other hand, CXCL12/CXCR4 chemokine axis may play an important role in localizing neoplasms to the niche. Thus, therapies that block the interaction between chemokine and chemokine receptors might prove to be efficacious therapeutic agents to prevent metastasis, as well.

Table 1.

Parallels between hematopoietic stem cells and metastatic neoplasms

Hematopoietic stem cells	Metastatic neoplasms
Hematopoietic stem cells	Leukemic/cancer stem cells
Unipotent	Undifferentiated
Homing	Metastasis
Colonization	Invasion
Quiescence	Dormancy
Migration	Dissemination
Self-renewal	Recurrence
Differentiation	Tumorigenesis

Molecular similarities between HSCs, LSCs and CSCs. The bone marrow stromal cells provide survival signals to HSCs, LSCs and CSCs probably through similar pathways. Yet, although a number of similarities exist between normal HSCs, LSCs and CSCs in terms of molecular mechanisms that regulate self-renewal and interactions with the bone marrow microenvironment, clearly differences exist as well.

Figure 3.

A model of the neoplastic bone metastasis (this figure was inspired by information found at: http://www.isrec.ch/research/groups/research_groups_detail_eid_1682_lid_2.htm; accessed 30 January 2008). The primary tumor becomes malignant and starts distributing tumor cells through the blood circulation to different organs. Chemokines such as SDF-1/CXC chemokine ligand 12 (CXCL12) may mediate the homing of neoplasm. Circulating neoplasm invades through the endothelial layer and basement membrane and reaches the secondary site or neoplastic niche. Once neoplasm localize to the marrow, those cells that are able to successfully engage the niche regulatory systems may become dormant through specific cell-to-cell adhesive interactions. In the mean time, those cells start grow again at secondary sites.

In this review, we have focused on the important role of bone marrow stromal cells in HSC, LSC and CSC niches. As described above, the roles of MSCs as HSC niche participants have also recently been realized. As such, HSCs and bone marrow stromal cells, including osteoblasts, endothelial cells and MSCs, are believed to localize closely in the bone marrow (Figure 1a). They are also thought to affect each other and the function of the niche. Therefore, osteoblasts, endothelial cells and MSCs are also likely to be involved in the LSC or CSC niche, albeit unwittingly (Figure 1b).

Further understanding of the interactions between HSCs and bone marrow stromal cells, including osteoblasts, endothelial cells and MSCs, might be useful for new therapeutic strategies to the bone marrow transplantation. Targeting drugs to supporting cells of the niche or adhesion and other molecules might also amplify the efficacy of traditional chemotherapy for leukemia and cancers.