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“Caught in the net”: the extracellular matrix of the bone marrow in normal hematopoiesis and leukemia

  • Costanza Zanetti
    Affiliations
    Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main, Germany
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  • Daniela S. Krause
    Correspondence
    Offprint requests to: Daniela S. Krause, Georg-Speyer-Haus Institute for Tumor Biology and Experimental Therapy, Paul-Ehrlich-Strasse 42-44, 60596 Frankfurt am Main, Germany
    Affiliations
    German Cancer Research Center (DKFZ), Heidelberg, Germany

    German Cancer Consortium (DKTK), Germany

    Frankfurt Cancer Institute, Frankfurt, Germany

    Faculty of Medicine, Johann Wolfgang Goethe University, Frankfurt, Germany
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Open AccessPublished:August 01, 2020DOI:https://doi.org/10.1016/j.exphem.2020.07.010

      Highlights

      • The ECM supports the HSC-supportive function of the bone marrow microenvironment.microenvironment
      • The ECM provides a scaffold and serves as a source of growth factors and proteases.
      • ECM proteins influence HSC function and the course of a leukemia.
      • Interactions between ECM proteins and HSCs or LSCs can be targeted therapeutically.
      • Novel technologies aid the understanding of the ECM in the BMM.
      The influence of the bone marrow microenvironment on normal hematopoiesis, but also leukemia, has largely been accepted. However, the focus has been predominantly on the role of various cell types or cytokines maintaining hematopoietic stem cells or protecting leukemia stem cells from different therapies. A frequently overlooked component of the bone marrow microenvironment is the extracellular matrix, which not only provides a mechanical scaffold, but also serves as a source of growth factors. We discuss here how extracellular matrix proteins directly or indirectly modulate hematopoietic stem cell physiology and influence leukemia progression. It is hoped that existing and future studies on this topic may propel forward the possibility of augmenting normal hematopoiesis and improving therapies for leukemia, for instance, by targeting of the extracellular matrix in the bone marrow.

      Hematopoiesis and leukemia

      After many decades of research, we can now postulate that hematopoiesis is a process in which hematopoietic stem cells (HSCs) are responsible for the production of mature differentiated blood cells via intermediate progenitor cells, but at the same time HSCs self-renew to maintain their own pool. Both processes occur through the regulation of key genes and defined genetic programs [
      • Riddell J
      • Gazit R
      • Garrison BS
      • et al.
      Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors.
      ]. To maintain a steady state, most HSCs remain in quiescence, which has been linked to long-term reconstitution capacity. However, HSCs rapidly switch to active proliferation under stress conditions [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ] and during differentiation [
      • Passegue E
      • Wagers AJ
      • Giuriato S
      • Anderson WC
      • Weissman IL
      Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates.
      ].
      Overall, studies on hematopoiesis and HSCs have also provided the basis for the “cancer stem cell” theory, that is, cancer stem cells represent a small, functionally distinct subset of tumor cells, which have the ability to reconstitute the tumor because of their ability to self-renew and differentiate into the various cancerous cells. The bulk tumor cells, however, do not have this ability.
      Leukemias are blood cancers characterized by abnormalities of leukocytes. The concept of leukemia stem cells (LSCs) was introduced when it was found that CD34+CD38− cells from acute myeloid leukemia (AML) patients have leukemia-initiating capacity upon transplantation into immunosuppressed mice [
      • Bonnet D
      • Dick JE.
      Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
      ]. Therefore, a leukemia was proven to be composed of heterogeneous cells, with only a subset of them having self-renewal capacity similar to that of normal HSCs. Complexity has been added to this question by the discovery of the contributions of the bone marrow microenvironment (BMM) or niche to the development, progression, and therapy resistance of leukemia.

      Bone marrow microenvironment

      The term stem cell niche, referring to a specific location and entity extrinsically orchestrating and controlling the self-renewal and differentiation capacity of HSCs, was coined in 1978 [
      • Schofield R.
      The relationship between the spleen colony-forming cell and the haemopoietic stem cell.
      ]. The BMM has largely been recognized as a unique structure providing a complex cellular, chemical, and mechanical microenvironment [
      • Morrison SJ
      • Scadden DT.
      The bone marrow niche for haematopoietic stem cells.
      ,
      • Birbrair A
      • Frenette PS.
      Niche heterogeneity in the bone marrow.
      ,
      • Crane GM
      • Jeffery E
      • Morrison SJ
      Adult haematopoietic stem cell niches.
      ]. At first, in the early years of the 21st century, the BMM was somewhat formally divided into endosteal [
      • Calvi LM
      • Adams GB
      • Weibrecht KW
      • et al.
      Osteoblastic cells regulate the haematopoietic stem cell niche.
      ,
      • Zhang J
      • Niu C
      • Ye L
      • et al.
      Identification of the haematopoietic stem cell niche and control of the niche size.
      ,
      • Taichman R
      • Reilly M
      • Verma R
      • Ehrenman K
      • Emerson S
      Hepatocyte growth factor is secreted by osteoblasts and cooperatively permits the survival of haematopoietic progenitors.
      ,
      • Nilsson SK
      • Johnston HM
      • Coverdale JA
      Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches.
      ] and vascular [
      • Kiel MJ
      • Yilmaz OH
      • Iwashita T
      • Yilmaz OH
      • Terhorst C
      • Morrison SJ
      SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.
      ,
      • Kusumbe AP
      • Ramasamy SK
      • Adams RH
      Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone.
      ,
      • Kunisaki Y
      • Bruns I
      • Scheiermann C
      • et al.
      Arteriolar niches maintain haematopoietic stem cell quiescence.
      ] niches. Since then, via the generation of a myriad of transgenic mouse models [
      • Ding L
      • Morrison SJ
      Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches.
      ,
      • Mendez-Ferrer S
      • Michurina TV
      • Ferraro F
      • et al.
      Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.
      ,
      • Mizoguchi T
      • Pinho S
      • Ahmed J
      • et al.
      Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development.
      ,
      • Ding L
      • Saunders TL
      • Enikolopov G
      • Morrison SJ
      Endothelial and perivascular cells maintain haematopoietic stem cells.
      ], sophisticated imaging technologies [
      • Mendez-Ferrer S
      • Michurina TV
      • Ferraro F
      • et al.
      Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.
      ,
      • Itkin T
      • Gur-Cohen S
      • Spencer JA
      • et al.
      Distinct bone marrow blood vessels differentially regulate haematopoiesis.
      ,
      • Hawkins ED
      • Duarte D
      • Akinduro O
      • et al.
      T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments.
      ,
      • Duarte D
      • Hawkins ED
      • Akinduro O
      • et al.
      Inhibition of endosteal vascular niche remodeling rescues hematopoietic stem cell loss in AML.
      ,
      • Lo Celso C
      • Fleming HE
      • Wu JW
      • et al.
      Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.
      ], and single-cell studies [
      • Tikhonova AN
      • Dolgalev I
      • Hu H
      • et al.
      The bone marrow microenvironment at single-cell resolution.
      ,
      • Baccin C
      • Al-Sabah J
      • Velten L
      • et al.
      Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization.
      ,
      • Wolock SL
      • Krishnan I
      • Tenen DE
      • et al.
      Mapping distinct bone marrow niche populations and their differentiation paths.
      ], the field has come to the understanding that the BMM is a continuum in which specific locations may be associated with specific features of HSCs [
      • Calvi LM
      • Adams GB
      • Weibrecht KW
      • et al.
      Osteoblastic cells regulate the haematopoietic stem cell niche.
      ,
      • Kunisaki Y
      • Bruns I
      • Scheiermann C
      • et al.
      Arteriolar niches maintain haematopoietic stem cell quiescence.
      ,
      • Itkin T
      • Gur-Cohen S
      • Spencer JA
      • et al.
      Distinct bone marrow blood vessels differentially regulate haematopoiesis.
      ,
      • Lo Celso C
      • Wu JW
      • Lin CP
      In vivo imaging of hematopoietic stem cells and their microenvironment.
      ,
      • Sugiyama T
      • Kohara H
      • Noda M
      • Nagasawa T
      Maintenance of the hematopoietic stem cell pool by CXCL12–CXCR4 chemokine signaling in bone marrow stromal cell niches.
      ,
      • Cordeiro Gomes A
      • Hara T
      • Lim VY
      • et al.
      Hematopoietic stem cell niches produce lineage-instructive signals to control multipotent progenitor differentiation.
      ,
      • Tokoyoda K
      • Egawa T
      • Sugiyama T
      • Choi BI
      • Nagasawa T
      Cellular niches controlling B lymphocyte behavior within bone marrow during development.
      ,
      • Zhao M
      • Perry JM
      • Marshall H
      • et al.
      Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells.
      ] and LSCs [
      • Bowers M
      • Zhang B
      • Ho Y
      • Agarwal P
      • Chen CC
      • Bhatia R
      Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development.
      ,
      • Ishikawa F
      • Yoshida S
      • Saito Y
      • et al.
      Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region.
      ]. In general, it is understood that the BMM is a complex entity composed of several cellular and acellular elements such as mesenchymal stromal cells (MSCs), endothelial cells (ECs), osteoblasts, osteoclasts, osteocytes, adipocytes, C–X–C motif chemokine ligand (CXCL)12-abundant reticular (CAR) cells, fibroblasts, sympathetic neurons, megakaryocytes, and macrophages, as well as chemical and mechanical factors and the extracellular matrix (ECM) [
      • Mendez-Ferrer S
      • Bonnet D
      • Steensma DP
      • et al.
      Bone marrow niches in haematological malignancies.
      ]. In addition, a specific pH, an oxygen gradient, cytokines, and mechanical factors, most of which are reviewed elsewhere [
      • Schepers K
      • Campbell TB
      • Passegue E
      Normal and leukemic stem cell niches: insights and therapeutic opportunities.
      ], contribute to the BMM's unique features (Figure 1). This review, however, is focused on the structure and biochemical properties of the proteins in the ECM of the BMM, as well as their functions in normal and malignant hematopoiesis.
      Figure 1
      Figure 1Hematopoietic stem cells and the bone marrow microenvironment. The (BMM) is composed of several cellular and acellular components such as osteoblasts, osteoclasts, adipocytes, sympathetic neurons, macrophages, mesenchymal stromal cells, pericytes, endothelial cells, CAR cells, the extracellular matrix, chemokines, and growth factors. Dynamic interactions between HSCs and the BMM, including oxygen concentration, pH, and mechanical factors, regulate HSC fate.

      The extracellular matrix in the bone marrow microenvironment

      In general, the ECM is a three-dimensional, complex and highly dynamic noncellular network composed of (1) proteoglycans; (2) fibrous proteins such as collagens, fibronectins, elastins, tenascins, vitronectin, and laminins; and (3) glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate, heparan sulfate, keratan sulfate, dermatan sulfate, and heparin; and (4) matricellular proteins such as osteocalcin and periostin, which assist in the linkage between other ECM proteins and cellular receptors [
      • Coutu DL
      • Kokkaliaris KD
      • Kunz L
      • Schroeder T
      Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules.
      ,
      • Klamer S
      • Voermans C.
      The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment.
      ]. The most abundant proteins in the ECM of the BMM are fibronectin, collagens I, II, III, IV, and X, laminin, tenascin, thrombospondin, and elastin. Soluble or membrane-bound glycoproteins of the sialomucin family, such as platelet–selectin glycoprotein ligand (PSGL1/CD162) and intercellular adhesion molecule 1 (ICAM1; CD54), are also associated with the ECM [
      • Klamer S
      • Voermans C.
      The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment.
      ]. The ECM regulates structural scaffolding and, here in particular, the stiffness and deformability of tissues and tissue homeostasis. It also serves as a major source of proteases and growth factors [
      • Hynes RO.
      The extracellular matrix: not just pretty fibrils.
      ].
      Possibly consistent with the early discovery that localization within the BM cavity may lead to differences in the proliferation rate of colony-forming units [
      • Lord BI
      • Testa NG
      • Hendry JH
      The relative spatial distributions of CFUs and CFUc in the normal mouse femur.
      ], the distribution of ECM components in the BMM has been reported to be spatially organized [
      • Nilsson SK
      • Debatis ME
      • Dooner MS
      • Madri JA
      • Quesenberry PJ
      • Becker PS
      Immunofluorescence characterization of key extracellular matrix proteins in murine bone marrow in situ.
      ]. In particular, collagens I and III, osteocalcin, vitronectin, osteopontin, and periostin were found predominantly in the bone matrix. Collagen IV and laminin were localized largely in the vascular basement membrane and the bone marrow parenchyma, while fibronectin was found only in the latter. The heparan sulfate proteoglycan perlecan was found in the bone matrix and arteriolar basement membrane [
      • Coutu DL
      • Kokkaliaris KD
      • Kunz L
      • Schroeder T
      Three-dimensional map of nonhematopoietic bone and bone-marrow cells and molecules.
      ]. Hyaluronan (HA) was highly expressed in the endothelium of blood vessels in the metaphysis, where it may be involved in the homing of hematopoietic stem and progenitor cells (HSPCs) [
      • Ellis SL
      • Grassinger J
      • Jones A
      • et al.
      The relationship between bone, hemopoietic stem cells, and vasculature.
      ].
      ECM composition and remodeling vary from tissue to tissue, leading to biochemical and biophysical properties that specifically reflect each environment [
      • Mouw JK
      • Ou G
      • Weaver VM
      Extracellular matrix assembly: a multiscale deconstruction.
      ]. Deregulation of components of the ECM can contribute to several pathological conditions such as cardiomyopathy [
      • Pauschinger M
      • Knopf D
      • Petschauer S
      • et al.
      Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio.
      ], fibrosis [
      • Walraven M
      • Hinz B
      Therapeutic approaches to control tissue repair and fibrosis: extracellular matrix as a game changer.
      ,
      • Bhattacharyya S
      • Tamaki Z
      • Wang W
      • et al.
      FibronectinEDA promotes chronic cutaneous fibrosis through Toll-like receptor signaling.
      ], and cancers [
      • Butcher DT
      • Alliston T
      • Weaver VM
      A tense situation: forcing tumour progression.
      ,
      • Kessenbrock K
      • Plaks V
      • Werb Z
      Matrix metalloproteinases: regulators of the tumor microenvironment.
      ]. In particular, mutations in the COL1A1 and COL1A2 genes, encoding the two α chains of type I collagen, cause autosomal dominant osteogenesis imperfecta. This results in abnormal synthesis and secretion of collagen type I, leading to bone fragility and deformities in patients [
      • Pollitt R
      • McMahon R
      • Nunn J
      • et al.
      Mutation analysis of COL1A1 and COL1A2 in patients diagnosed with osteogenesis imperfecta type I–IV.
      ]. In addition, mutations in the COL7A1 and COL17A1 genes are responsible for a dystrophic form of epidermolysis bullosa [
      • Pasmooij AM
      • Garcia M
      • Escamez MJ
      • et al.
      Revertant mosaicism due to a second-site mutation in COL7A1 in a patient with recessive dystrophic epidermolysis bullosa.
      ], and mutations in the COL5A1 or COL5A2 genes give rise to classic Ehlers–Danlos syndrome, a rare autosomal dominant connective tissue disorder [
      • Ritelli M
      • Dordoni C
      • Venturini M
      • et al.
      Clinical and molecular characterization of 40 patients with classic Ehlers–Danlos syndrome: identification of 18 COL5A1 and 2 COL5A2 novel mutations.
      ].

      The extracellular matrix and hematopoiesis

      ECM proteins are important constituents of the BM milieu, where they are involved in adhesion, binding of cytokines, and the general compartmentalization of the BM [
      • Klein G.
      The extracellular matrix of the hematopoietic microenvironment.
      ] (Figure 2). The fact that ECM proteins can be extracted and purified from different animal sources and used widely for the coating of cell culture wells has formed the basis for studies of the interactions of HSCs with the ECM. In addition, novel technologies, as mentioned below, have provided insight into the biochemical-functional properties of the ECM.
      Figure 2
      Figure 2Hematopoietic stem cells and the extracellular matrix. Extracellular matrix proteins are important components of the BMM and directly or indirectly modulate HSC proliferation, quiescence, migration, and adhesion. In addition, they provide a mechanical scaffold, which also serves as a source of growth factors and chemokines, thereby maintaining HSCs and homeostasis of differentiated cells. Specific and additional details are provided in the text. LN=laminin; VLA-4/5=very late antigen-4/5; MIP-1α=macrophage inflammatory protein-1α; PTH=parathyroid hormone; LMPP=lymphoid-primed multipotent progenitor cells; 5-FU=5-fluorouracil.
      Changes in the cellular microenvironment, in general, are sensed by cell–ECM interactions and are mediated mainly by integrins [
      • Kechagia JZ
      • Ivaska J
      • Roca-Cusachs P
      Integrins as biomechanical sensors of the microenvironment.
      ] as well as discoidin domain receptors (DDRs) [
      • Leitinger B
      • Hohenester E.
      Mammalian collagen receptors.
      ], leukocyte-associated Ig-like receptor (LAIR) 1 [
      • Meyaard L.
      The inhibitory collagen receptor LAIR-1 (CD305).
      ], and syndecans [
      • Morgan MR
      • Humphries MJ
      • Bass MD
      Synergistic control of cell adhesion by integrins and syndecans.
      ], regulating cell migration, adhesion, survival, shape, and differentiation [
      • Bissell MJ
      • Hall HG
      • Parry G
      How does the extracellular matrix direct gene expression?.
      ]. Integrins are heterodimers, consisting of an α subunit and a β subunit, of which there are 18 and 8, respectively. Integrins span the cell membrane with a usually short cytoplasmic domain, while the N-terminal domain represents the binding region for ligands from the ECM. Murine HSPCs express integrin α4β1 (VLA4; ITGA4 and ITGB1), α5β1 (VLA5; ITGA5 and ITGB1), α6β1, αLβ2 (LFA1; ITGAL and ITGB2), and αMβ2 (MAC-1; ITGAM and ITGB2), while some HSPCs express integrin 3 (ITGB3) [
      • Klamer S
      • Voermans C.
      The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment.
      ]. Binding of integrins to proteins of the ECM, also termed outside-in signaling "outside-in signaling", activates intracellular signaling pathways leading to modulation of the position of other membrane receptors, the actin cytoskeleton, and possibly the cell cycle. This regulates cell spreading and retraction, migration, survival, shape, and differentiation. “Inside-out signaling,” that is, cell-intrinsic processes, which may lead to clustering or conformational changes of integrins, in contrast, stimulates ligand binding by integrins [
      • Calderwood DA
      Integrin activation.
      ] and regulates integrin adhesiveness. This may be stimulated in normal and leukemic CD34+ HSCs by growth factors [
      • Lévesque JP
      • Leavesley DI
      • Niutta S
      • Vadas M
      • Simmons PJ
      Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.
      ,
      • Moore S
      • Haylock DN
      • Lévesque JP
      • et al.
      Stem cell factor as a single agent induces selective proliferation of the Philadelphia chromosome positive fraction of chronic myeloid leukemia CD34(+) cells.
      ]. β1 integrins are essential for the homing and colonization of HSPCs in the fetal liver and the bone marrow. α4 integrins are involved in the differentiation of erythroid, myeloid, and B-cell progenitors, likely because of their role in transmigration of HSPCs through fibronectin. Humans and mice with mutations in the β2 integrin gene have leukocyte adhesion deficiency type I, leading to significant leukocytosis [
      • Bouvard D
      • Brakebusch C
      • Gustafsson E
      • et al.
      Functional consequences of integrin gene mutations in mice.
      ]. Expression of DDR1 is found in epithelial cells, while DDR2 is expressed in MSCs and immature dendritic cells [
      • Gonzalez ME
      • Martin EE
      • Anwar T
      • et al.
      Mesenchymal stem cell-induced DDR2 mediates stromal–breast cancer interactions and metastasis growth.
      ,
      • Borza CM
      • Pozzi A.
      Discoidin domain receptors in disease.
      ]. LAIR-1, a receptor playing an inhibitory role in immune cell activation, is expressed on peripheral blood leukocytes and CD34+ HSCs [
      • Verbrugge A
      • de Ruiter T
      • Geest C
      • Coffer PJ
      • Meyaard L
      Differential expression of leukocyte-associated Ig-like receptor-1 during neutrophil differentiation and activation.
      ]. LAIR-1 knockout mice are characterized by increased numbers of splenic B, regulatory T, and dendritic cells [
      • Tang X
      • Tian L
      • Esteso G
      • et al.
      Leukocyte-associated Ig-like receptor-1-deficient mice have an altered immune cell phenotype.
      ].
      Several pieces of evidence for the involvement of the ECM in hematopoiesis exist. For example, deletion of Ext1, the gene encoding a glycosyltransferase essential for heparan sulfate production in BM stromal cells, led to mobilization of HSPCs from the BM [
      • Saez B
      • Ferraro F
      • Yusuf RZ
      • et al.
      Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning.
      ]. Further, three-dimensional quantitative microscopy has revealed that sinusoidal endothelial cells and mesenchymal CXCL-12–abundant reticular cells closely associate with the ECM [
      • Gomariz A
      • Helbling PM
      • Isringhausen S
      • et al.
      Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy.
      ]. With the use of microarray equipment, ECM ligands have been deposited on a substrate and used to study neural stem cell self-renewal and differentiation in a high-throughput assay, a strategy that can easily be adopted by the hematopoiesis field [
      • Soen Y
      • Mori A
      • Palmer TD
      • Brown PO
      Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.
      ].
      Among the growth factors stored in the ECM are fibroblast growth factors (FGFs), interleukin (IL)-1, transforming growth factor (TGF) β1, and others, which may be secreted by BM stromal cells or hematopoietic precursors and influence hematopoietic lineage specification and proliferation [
      • Bodo M
      • Baroni T
      • Tabilio A
      Haematopoietic and stromal stem cell regulation by extracellular matrix components and growth factors.
      ]. Calcium ions trapped in the ECM close to the endosteum, whose concentration at this location is thought to be 20-fold higher than in serum [
      • Silver IA
      • Murrills RJ
      • Etherington DJ
      Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts.
      ], also influence the localization and function of HSCs [
      • Adams GB
      • Chabner KT
      • Alley IR
      • et al.
      Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.
      ]. Further, divalent cations, for example, Ca2+, Mg2+, and Mn2+, in the ECM modify the affinity of β1 integrins on HSPCs to their ligands, such as vascular cell adhesion molecule (VCAM) 1 [
      • Chigaev A
      • Zwartz G
      • Graves SW
      • et al.
      Alpha4beta1 integrin affinity changes govern cell adhesion.
      ], regulating HSPC homing in combination with mitogenic cytokines like granulocyte–macrophage colony-stimulating factor and KIT ligand [
      • Takamatsu Y
      • Simmons PJ
      • Lévesque JP
      Dual control by divalent cations and mitogenic cytokines of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity expressed by human hemopoietic cells.
      ]. Other ions in the BMM are zinc and copper [
      • Domingues MJ
      • Cao H
      • Heazlewood SY
      • Cao B
      • Nilsson SK
      Niche extracellular matrix components and their influence on HSC.
      ]. Lastly, prostaglandin E2, produced by osteoblasts and derived from the eicosanoid family, sphingosine 1-phosphate, and other lipids are also considered regulators of HSC homeostasis.

      Components of the ECM and their role in hematopoiesis

      Collagen

      The most abundant ECM protein in the bone marrow is collagen type I, although collagen types II, III, V, and XI, as well as proteoglycans, contribute to bone structure and micro-architecture determining bone strength [
      • Tzaphlidou M.
      The role of collagen in bone structure: an image processing approach.
      ]. The structural hallmarks of all collagens are three individual α chains that form a triple-helical structure, which can be homotrimeric or heterotrimeric, depending on the 28 forms of collagen present [
      • Ricard-Blum S.
      The collagen family.
      ]. Collagen biosynthesis is a highly complex, multistep process, which occurs via chain association and folding of soluble precursors called pro-collagens and subsequent posttranslational modifications [
      • Kadler KE
      • Holmes DF
      • Trotter JA
      • Chapman JA
      Collagen fibril formation.
      ]. The pro-α chains constituting the collagen triple helix consist of the repetitive motif glycine (Gly)–X–Y [
      • Ramshaw JA
      • Shah NK
      • Brodsky B
      Gly-X-Y tripeptide frequencies in collagen: a context for host–guest triple-helical peptides.
      ]. The α chains undergo hydroxylation and glycosylation steps in the endoplasmic reticulum, resulting in the generation of the triple-helical pro-collagen molecule. Subsequently, lysine residues are de-aminated by lysyl oxidase (LOX), leading to the formation of cross-links between collagen molecules and collagen and elastin fibers, which contribute to tissue stiffness [
      • Oxlund H
      • Barckman M
      • Ortoft G
      • Andreassen TT
      Reduced concentrations of collagen cross-links are associated with reduced strength of bone.
      ]. As mentioned above, integrins, DDRs, and LAIR-1 recognize triple-helical collagen, thereby contributing to the homeostasis of the ECM [
      • Leitinger B
      • Hohenester E.
      Mammalian collagen receptors.
      ].
      In functional studies, collagen VI was a substrate for cyto-adhesion by various hematopoietic cell types [
      • Klein G
      • Müller CA
      • Tillet E
      • Chu ML
      • Timpl R
      Collagen type VI in the human bone marrow microenvironment: a strong cytoadhesive component.
      ]. Normal myeloid progenitors, mature peripheral blood neutrophils, monocytes, and burst-forming units erythroid exhibited significant binding to collagen I [
      • Koenigsmann M
      • Griffin JD
      • DiCarlo J
      • Cannistra SA
      Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I.
      ]. In collagen IXα1-deficient mice, disorganization of the trabecular bone, as well as increased fibronectin, was observed. Hematologically, myeloid cells exhibited reduced number and differentiation, and the immune response to certain bacteria was impaired in these mice [
      • Probst K
      • Stermann J
      • von Bomhard I
      • et al.
      Depletion of collagen IX alpha1 impairs myeloid cell function.
      ].

      Fibronectin

      The glycoprotein fibronectin (FN) is a key player in normal adhesion, migration differentiation, wound healing, and growth. FN is also essential for embryonic development, as homozygously mutant mice display early embryonic lethality from defects in mesodermally derived tissues, the neural tube, yolk sac, amnion, and vasculature [
      • George EL
      • Georges-Labouesse EN
      • Patel-King RS
      • Rayburn H
      • Hynes RO
      Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin.
      ]. In vertebrates, two major forms of FN are found: soluble or plasma fibronectin, which is synthesized by hepatocytes [
      • Tamkun JW
      • Hynes RO.
      Plasma fibronectin is synthesized and secreted by hepatocytes.
      ] and secreted into the blood, and insoluble cellular fibronectin, which is produced primarily by fibroblasts, chondrocytes, and macrophages [
      • Mao Y
      • Schwarzbauer JE
      Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.
      ].
      FN is encoded by an 8-kb mRNA. However, alternative splicing at three different sites of the pre-mRNA leads to 20 different isoforms of human FN. FN is a protein dimer composed of two almost identical 230- to 270-kDa monomers linked by a pair of disulfide bonds stabilizing the folded structure. Each monomer contains module types I, II, and III, whereby each module mediates interactions with FN itself, other components of the ECM, and cell surface receptors [
      • Mao Y
      • Schwarzbauer JE
      Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.
      ].
      The FN receptors belong to the integrin family of transmembrane adhesion receptors, which mediate cell–cell but also cell–ECM interactions. FN is secreted as a dimer, but FN assembly is dependent on cell contact and is mediated by various functional and protein-binding domains within the fibronectin monomer. The RGD sequence (Arg–Gly–Asp) represents the site of cell attachment of α5β1 and α5β3 integrins [
      • Mao Y
      • Schwarzbauer JE
      Fibronectin fibrillogenesis, a cell-mediated matrix assembly process.
      ]. FN-Mmediated tissue stiffness, matrix composition, and growth factor availability in the ECM activate outside-in signaling by integrins. Via various intermediate signaling proteins such as phosphoinositide 3-kinases (PI3Ks), the proto-oncogene tyrosine–protein kinase Src, and focal adhesion kinase (FAK), integrin-mediated signaling leads to stimulation of the Akt, ERK, Jnk, RhoA, and Rac1/Cdc42 pathways and alteration of cellular survival, proliferation, differentiation, migration, adhesion, and polarity [
      • Legate KR
      • Wickstrom SA
      • Fassler R
      Genetic and cell biological analysis of integrin outside-in signaling.
      ]. Deletion of the receptor tyrosine kinase FAK in hematopoietic cells led to an increase in cycling HSCs, possibly via an altered interaction with the BMM [
      • Lu J
      • Sun Y
      • Nombela-Arrieta C
      • et al.
      Fak depletion in both hematopoietic and nonhematopoietic niche cells leads to hematopoietic stem cell expansion.
      ].
      Fibronectin provides anchorage for HSC, which bind to the C-terminal, heparin-binding fragment of fibronectin via the α4 subunit of the very late antigen (VLA)-4 integrin receptor [
      • Williams DA
      • Rios M
      • Stephens C
      • Patel VP
      Fibronectin and VLA-4 in haematopoietic stem cell–microenvironment interactions.
      ]. In addition, HSPC bind via VLA-5 [
      • van der Loo JC
      • Xiao X
      • McMillin D
      • Hashino K
      • Kato I
      • Williams DA
      VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin.
      ]. Increased adhesion of human cord blood CD34+ progenitor cells to fibronectin was observed during transit through S phase of the cell cycle, while adhesion to vascular cell adhesion molecule 1 (VCAM-1) was decreased [
      • Huygen S
      • Giet O
      • Artisien V
      • Di Stefano I
      • Beguin Y
      • Gothot A
      Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture.
      ]. This suggests that binding to the ECM is accompanied by cell cycle changes in HSCs. Also, the immobilization of chemokines such as C–X–C motif chemokine ligand 12 (CXCL12) by its binding to heparan sulfate heavily influences HSC function [
      • Rueda P
      • Richart A
      • Recalde A
      • et al.
      Homeostatic and tissue reparation defaults in mice carrying selective genetic invalidation of CXCL12/proteoglycan interactions.
      ].

      Laminin

      Laminins (LNs) are high-molecular-weight ECM proteins and an integral part of basal lamina. Laminins are heterotrimers, consisting of α, β, and γ polypeptide chains, providing a structural scaffold. The crosslike structure of laminin promotes binding of other laminin molecules and the generation of laminin sheets. Fifteen laminin trimers have been identified, but LN-2, LN-8, and LN-10 are the predominant laminins in BM [
      • Gu Y
      • Sorokin L
      • Durbeej M
      • Hjalt T
      • Jonsson JI
      • Ekblom M
      Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells.
      ]. Laminins associate with FN and other ECM proteins in networks and bind to integrins on cellular surfaces. The matrix assembly of LN-5 is mediated by the integrin receptors α3β1 and α6β4 and signaling via the Rho, Rac1 and Cdc42 family of GTPases [
      • DeHart GW
      • Jones JC
      Myosin-mediated cytoskeleton contraction and Rho GTPases regulate laminin-5 matrix assembly.
      ].
      Laminin-10/11 contribute as adhesive substrates for CD34+ and CD34+CD38– HSPCs via an integrin α6 and β1 receptor-mediated mechanism. Laminin-10/11 were particularly adhesive to myelomonocytic and erythroid progenitor cells and several lymphoid and myeloid cell lines. Laminin-10/11 and laminin-8 also facilitated the migration of CD34+ cells stimulated by stromal-derived factor 1α (SDF-1α) [
      • Gu YC
      • Kortesmaa J
      • Tryggvason K
      • et al.
      Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells.
      ]. Laminin-421, which consists of laminin α4, β2, γ1 chains, is found predominantly around venous sinuses in a fibrous network in close association with HSPCs. Transplantation of BM from mice deficient for laminin subunit α4 (Lama4) into wild-type mice led to inefficient reconstitution, and BM from Lama4 knockout mice exhibited decreased colony formation in vitro. This was found to result from a reduced number of HSPCs in Lama4 knockout mice. In fact, Lin–c-kit+Sca1+CD48– HSCs from Lama4-null mice are retained in the G0 phase of the cell cycle and, therefore, are a more quiescent phenotype [
      • Susek KH
      • Korpos E
      • Huppert J
      • et al.
      Bone marrow laminins influence hematopoietic stem and progenitor cell cycling and homing to the bone marrow.
      ].

      Osteopontin

      Osteopontin (OPN) is a member of the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family of proteins [
      • Fisher LW
      • Torchia DA
      • Fohr B
      • Young MF
      • Fedarko NS
      Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin.
      ]. OPN, an important component of the bone matrix produced by osteolineage cells, MSCs, and other cells, is predominantly known as a secreted protein [
      • Zohar R
      • Lee W
      • Arora P
      • Cheifetz S
      • McCulloch C
      • Sodek J
      Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells.
      ,
      • Inoue M
      • Shinohara ML
      Intracellular osteopontin (iOPN) and immunity.
      ]. OPN exists in different isoforms as it undergoes alternative splicing, as well as posttranslational modifications [
      • Kazanecki CC
      • Uzwiak DJ
      • Denhardt DT
      Control of osteopontin signaling and function by post-translational phosphorylation and protein folding.
      ]. The protein interacts with α5β1, αvβ3, αvβ5, α9β1, and α4β1 integrin receptors [
      • Xie Y
      • Sakatsume M
      • Nishi S
      • Narita I
      • Arakawa M
      • Gejyo F
      Expression, roles, receptors, and regulation of osteopontin in the kidney.
      ], as well CD44 protein [
      • Weber GF
      • Ashkar S
      • Glimcher MJ
      • Cantor H
      Receptor–ligand interaction between CD44 and osteopontin (Eta-1).
      ]. OPN has a pleiotropic role as it is involved in cell survival, adhesion, migration, bone remodeling, angiogenesis, wound healing [
      • Standal T
      • Borset M
      • Sundan A
      Role of osteopontin in adhesion, migration, cell survival and bone remodeling.
      ,
      • Denhardt DT
      • Noda M.
      Osteopontin expression and function: role in bone remodeling.
      ], the stress response, and immune regulation [
      • Wang KX
      • Denhardt DT.
      Osteopontin: role in immune regulation and stress responses.
      ] and inflammation [
      • Lund SA
      • Giachelli CM
      • Scatena M
      The role of osteopontin in inflammatory processes.
      ].
      Mice deficient for osteopontin are characterized by an increased number and reduced apoptosis of HSCs. Stimulation of the microenvironment with parathyroid hormone in the absence of osteopontin led to a further increase in HSC numbers, suggesting that osteopontin is a negative regulator of HSC function and number, especially in the setting of stress [
      • Stier S
      • Ko Y
      • Forkert R
      • et al.
      Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size.
      ].
      Lack of osteopontin in aged mice was associated with increased lymphocytes and decreased erythrocytes in peripheral blood. Aged osteopontin-deficient HSCs exhibited decreased reconstitution ability, for which impaired differentiation was considered causative. Severe anemia, thrombocytopenia, and increased deaths were observed in serial transplantation experiments of aged osteopontin-deficient compared with wild-type BM [
      • Li J
      • Carrillo Garcia C
      • Riedt T
      • et al.
      Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging.
      ]. Osteopontin also contributes to the migration of HSCs toward the endosteum, the adhesion of HSPCs via β1 integrins, and their decreased cycling [
      • Nilsson SK
      • Johnston HM
      • Whitty GA
      • et al.
      Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.
      ].

      Tenascin-C

      Tenascin-C (TN-C) is a glycoprotein consisting of several polypeptides of 180–300 kDa each. TN-C is highly conserved among vertebrates [
      • Midwood KS
      • Chiquet M
      • Tucker RP
      • Orend G
      Tenascin-C at a glance.
      ]. It is found in the ECM, where it is expressed during development, disease, or injury. During development, TN-C plays a morphoregulatory role, but in the BM it has specific adhesive functions for HSCs [
      • Klein G
      • Beck S
      • Muller CA
      Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment.
      ]. Tenascins share a similar structure, as they contain heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domains, and a C-terminal globular domain. The N-terminus of TN-C contains an oligomerization domain, at which TN-C is formed into hexamers. TN-C (and TN-R) exist in different isoforms, and TN-C is known to interact with fibronectin. Tenascin, via its EGF-like domain, has been implicated in cellular growth and differentiation [
      • Engel J.
      EGF-like domains in extracellular matrix proteins: localized signals for growth and differentiation?.
      ]; however, its role in cell adhesion is controversial, as it seems dependent on the splicing variant, as well as the binding substrate [
      • Ghert MA
      • Qi WN
      • Erickson HP
      • Block JA
      • Scully SP
      Tenascin-C splice variant adhesive/anti-adhesive effects on chondrosarcoma cell attachment to fibronectin.
      ].
      In TN-C-deficient mice, although developmentally normal, the colony-forming capacity of BM cells was shown to be reduced [
      • Ohta M
      • Sakai T
      • Saga Y
      • Aizawa S
      • Saito M
      Suppression of hematopoietic activity in tenascin-C-deficient mice.
      ]. Their BM architecture and mononuclear cell count, however, did not differ from those of wild-type mice. However, in various in vitro assays, defects were observed in the production of hematopoietic cells, colony-forming capacity, and longevity of cultures of BM cells from TN-C-deficient mice. In contrast, addition of TN-C to these cultures rescued this effect. In line with this, TN-C was reported to enhance HSC proliferation [
      • Seiffert M
      • Beck SC
      • Schermutzki F
      • Muller CA
      • Erickson HP
      • Klein G
      Mitogenic and adhesive effects of tenascin-C on human hematopoietic cells are mediated by various functional domains.
      ]. TN-C is increased after myeloablation, and TN-C-deficient mice had higher lethality rates and impairment of hematopoietic recovery after injection of 5-fluorouracil (5-FU) or sublethal irradiation. TN-C-deficient mice failed to support wild-type hematopoietic cells after transplantation [
      • Nakamura-Ishizu A
      • Okuno Y
      • Omatsu Y
      • et al.
      Extracellular matrix protein tenascin-C is required in the bone marrow microenvironment primed for hematopoietic regeneration.
      ]. Moreover, tenascin inhibits T-cell activation via T-cell receptor/CD3 [
      • Hibino S
      • Kato K
      • Kudoh S
      • Yagita H
      • Okumura K
      Tenascin suppresses CD3-mediated T cell activation.
      ]. Lack of TN-C in mice led to decreased numbers of T-cell progenitors in the thymus and a redistribution to other lymphoid organs, suggesting an adhesive role for TN-C also in lymphoid cells. TN-C was reported to be important for the retention and homing of lymphoid primed progenitor cells via interaction with integrin α9β1 [
      • Ellis SL
      • Heazlewood SY
      • Williams B
      • et al.
      The role of tenascin C in the lymphoid progenitor cell niche.
      ].

      Hyaluronan

      Hyaluronan or hyaluronic acid (HA) is a high-molecular-mass polysaccharide and a member of the glycosaminoglycans (GAGs). It is found in connective, epithelial, and neural tissues, as well as cartilage. HA is not formed in the Golgi apparatus as are other GAGs, but at the plasma membrane [
      • Laurent TC
      • Fraser JR.
      Hyaluronan.
      ]. HA, a major component of the ECM, plays an important role in cell proliferation, migration, and bone resorption [
      • Prince CW.
      Roles of hyaluronan in bone resorption.
      ]. Furthermore, exogenous administration of HA increased the proliferation of porcine BM stromal cells, as well as later gene expression of osteocalcin and calcium deposition in vitro [
      • Zou L
      • Zou X
      • Chen L
      • et al.
      Effect of hyaluronan on osteogenic differentiation of porcine bone marrow stromal cells in vitro.
      ]. The most prominent HA receptor is CD44 [
      • Aruffo A
      • Stamenkovic I
      • Melnick M
      • Underhill CB
      • Seed B
      CD44 is the principal cell surface receptor for hyaluronate.
      ,
      • Lesley J
      • Hascall VC
      • Tammi M
      • Hyman R
      Hyaluronan binding by cell surface CD44.
      ], and their interactions lead to inflammatory and tumorigenic responses [
      • Misra S
      • Hascall VC
      • Markwald RR
      • Ghatak S
      Interactions between hyaluronan and its receptors (CD44, RHAMM) regulate the activities of inflammation and cancer.
      ].

      Periostin

      Periostin, a secreted ECM protein produced by mesenchymal cells, regulates HSC proliferation via integrin-αv (Itgav). Deficiency of periostin in mice leads to anemia, myelomonocytosis, and lymphopenia in peripheral blood, although HSCs are increased in the BM. The recovery of the hematopoietic system after irradiation is faster in periostin knockout mice. The periostin–Itgav interaction leads to inhibition of the FAK/PI3K/AKT pathway, an increase in the expression of p27Kip1 in HSCs, and perpetuation of quiescent HSCs [
      • Khurana S
      • Schouteden S
      • Manesia JK
      • et al.
      Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin–Itgav axis.
      ]. In addition, it was recently found that the vitamin K antagonist and anticoagulant warfarin leads to an impairment of the binding of decarboxylated periostin to integrin β3 on HSPCs and, therefore, hematopoiesis [
      • Verma D
      • Kumar R
      • Pereira RS
      • et al.
      Vitamin K antagonism impairs the bone marrow microenvironment and hematopoiesis.
      ] (Figure 2).

      Other extracellular matrix proteins and hematopoiesis

      Arteriolar endothelial cells, CAR cells, and cells of the osteoblastic lineage express the secreted ECM protein Del-1. Del-1 increases myeloid bias of HSCs by promoting their proliferation and differentiation toward this lineage. This became particularly apparent during stress such as HSC transplantation, administration of granulocyte colony-stimulating factor, and inflammation. The effects of Del-1 were mediated via integrin β3 [
      • Mitroulis I
      • Chen LS
      • Singh RP
      • et al.
      Secreted protein Del-1 regulates myelopoiesis in the hematopoietic stem cell niche.
      ]. Dermatopontin is another non-collagenous protein of the ECM, which, in cooperation with other ECM proteins, promotes the adhesion of hematopoietic cells to plastic and stromal cells in vitro. However, dermatopontin-deficient mice did not differ from wild-type mice with respect to overall cellularity, HSC numbers, and colony-forming ability.
      The glycoprotein vitronectin belongs to the family of hemopexins and is found in the ECM, blood, and bone. Its binding to αVβ3 integrin augments cell adhesion, while its binding to plasminogen activation inhibitor 1 may regulate the proteolytic degradation of the ECM [
      • Schvartz I
      • Seger D
      • Shaltiel S
      Vitronectin.
      ].
      The selectin family of transmembrane proteins, namely, endothelial (E)-, platelet (P)- and leukocyte (L)-selectins, also play a role in cell–matrix (and cell–cell) interactions in the BMM via their binding to sialylated, fucosylated, or sulfated carbohydrate groups on glycoproteins [
      • Klamer S
      • Voermans C.
      The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment.
      ].
      Lastly, the ECM adaptor protein matrilin-4 (Matn4), which is expressed in HSCs, is downregulated under stress conditions. In fact, low levels of matrilin-4 and subsequent downregulation of CXCR4 increase HSPC numbers after transplantation or chemotherapy or in inflammatory conditions, suggesting that matrilin-4 is a negative regulator of the response of HSCs to stress [
      • Uckelmann H
      • Blaszkiewicz S
      • Nicolae C
      • et al.
      Extracellular matrix protein matrilin-4 regulates stress-induced HSC proliferation via CXCR4.
      ].

      Mechanical effects of the extracellular matrix on hematopoiesis

      One frequently overlooked role of the ECM in the BMM is its provision of a mechanical scaffold and elasticity. Upon culture on a tropoelastin-coated system, undifferentiated cells, including HSPCs, were expanded two- to threefold. This expansion was dependent on substrate elasticity, as truncated or non-cross-linked tropoelastin, as well as inhibited mechanotransduction, abolished these effects [
      • Holst J
      • Watson S
      • Lord MS
      • et al.
      Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells.
      ]. In addition, culture of CD34+ human HSCs from umbilical cord blood in collagen I fibrils in the presence of HSC-supportive cytokines led to an increase in colony-forming units compared with controls. Gene expression analysis in HSCs after culture in collagen fibrils revealed an upregulation of several growth factors, cytokines, and chemokines such as IL-8 and macrophage inhibitory protein 1α compared with controls [
      • Oswald J
      • Steudel C
      • Salchert K
      • et al.
      Gene-expression profiling of CD34+ hematopoietic cells expanded in a collagen I matrix.
      ]. Furthermore, it has been found that engagement of integrins and activation of myosin II in HSCs affect the proliferation and myeloid differentiation of murine HSCs, as tested by ECM ligand-coated polyacrylamide substrates of defined stiffness. Fibronectin, in combination with stiffness of the BM matrix, maintained hematopoietic progenitor cells, whereas collagen and laminin promoted proliferation and myeloid differentiation, respectively. Inhibition of integrins α5β1 and α1β3 abolished these effects [
      • Choi JS
      • Harley BA.
      Marrow-inspired matrix cues rapidly affect early fate decisions of hematopoietic stem and progenitor cells.
      ]. In drosophila, integrins regulate hematopoiesis and the postinfection immune response via organization of the ECM and its density via the bone morphogenetic protein (BMP) and Hedgehog (Hh) pathways [
      • Khadilkar RJ
      • Ho KYL
      • Venkatesh B
      • Tanentzapf G
      Integrins modulate extracellular matrix organization to control cell signaling during hematopoiesis.
      ].

      The extracellular matrix in hematological cancers and its possible targeting

      Integrins are also important receptors for the interaction of leukemia cells with the ECM. Dysfunctional β1 integrin plays a role in the altered adhesion of chronic myeloid leukemia (CML) cells to fibronectin, which is reversed by interferon α. Increased binding to VCAM-1 by AML cells via β1 integrins was associated with prolonged patient survival [
      • Krause DS
      • Scadden DT.
      A hostel for the hostile: the bone marrow niche in hematologic neoplasms.
      ], and treatment with an antibody to VLA-4, natalizumab, in a xenotransplantation model of AML led to improved survival [
      • Rashidi A
      • DiPersio JF.
      Targeting the leukemia–stroma interaction in acute myeloid leukemia: rationale and latest evidence.
      ]. β3 integrin is an important mediator of the progression of AML [
      • Miller PG
      • Al-Shahrour F
      • Hartwell KA
      • et al.
      In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling.
      ] and imatinib-resistant CML [
      • Kumar R
      • Pereira R
      • Zanetti C
      • et al.
      Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia.
      ].
      Some published reports have indicated that, indeed, targeting of the ECM in the LSC niche may lead to prolongation of survival of mice with leukemia. Increased bone remodeling resulting from treatment with parathyroid hormone, for example, led to a 15-fold reduction of LSCs in CML via the release of TGF-β1 from the ECM. However, the same did not hold true for AML, likely because of differences in the expression of TGF-β receptor I [
      • Krause DS
      • Fulzele K
      • Catic A
      • et al.
      Differential regulation of myeloid leukemias by the bone marrow microenvironment.
      ]. In addition, administration of fibronectin or inhibition of the pseudokinase integrin-linked kinase (ILK)—which is a component of the adhesosome, signals downstream of integrin β1 and β3, regulates fibronectin deposition and was reported to play a role in mediating resistance to tyrosine kinase inhibitors [
      • Rothe K
      • Babaian A
      • Nakamichi N
      • et al.
      Integrin-linked kinase mediates therapeutic resistance of quiescent CML stem cells to tyrosine kinase inhibitors.
      ]—can prolong survival in a murine model of imatinib-resistant CML [
      • Kumar R
      • Pereira R
      • Zanetti C
      • et al.
      Specific, targetable interactions with the microenvironment influence imatinib-resistant chronic myeloid leukemia.
      ]. Inhibition of FAK, another component of the adhesosome, which was more highly expressed in patients having an AML relapse, led to survival prolongation in an AML xenograft model [
      • Carter BZ
      • Mak PY
      • Wang X
      • et al.
      Focal adhesion kinase as a potential target in AML and MDS.
      ] and in BCR-ABL1+ B-ALL in combination with a tyrosin kinase inhibitor [
      • Churchman ML
      • Evans K
      • Richmond J
      • et al.
      Synergism of FAK and tyrosine kinase inhibition in Ph+ B-ALL.
      ].
      The role of the ECM has also been studied in lymphoid leukemias, where periostin was found to be highly expressed in the BM of patients with B-ALL. Additionally, transplantation of B-ALL cells into periostin-deficient mice significantly decreased leukemia burden [
      • Ma Z
      • Zhao X
      • Huang J
      • et al.
      A critical role of periostin in B-cell acute lymphoblastic leukemia.
      ]. A follow-up study found that a reciprocal interplay exists between BM and MSCs, which secrete periostin independent of CCL2 production by B-ALL cells. The secreted periostin promotes B-ALL progression via stimulation of the integrin–ILK–NF-κB pathway [
      • Ma Z
      • Zhao X
      • Deng M
      • et al.
      Bone marrow mesenchymal stromal cell-derived periostin promotes B-ALL progression by modulating CCL2 in leukemia cells.
      ]. Furthermore, we have recently found that B-ALL cells instruct the BMM via release of TNF-α, leading to increased production of matrix metalloproteinase (MMP)-9 by MSCs. Consequently, the ECM undergoes increased degradation and increased invasion by leukemia cells, whereby leukemia progression could be delayed via administration of an MMP-9 inhibitor [
      • Verma D
      • Zanetti C
      • Godavarthy PS
      • et al.
      Bone marrow niche-derived extracellular matrix-degrading enzymes influence the progression of B-cell acute lymphoblastic leukemia.
      ].
      Adhesion to ECM proteins such as osteopontin has also been found to influence the dormancy of B-ALL cells. Conversely, inhibition of osteopontin increased B-ALL cell proliferation and increased leukemia progression in mice and, in combination with chemotherapy, reduced minimal residual disease [
      • Boyerinas B
      • Zafrir M
      • Yesilkanal AE
      • Price TT
      • Hyjek EM
      • Sipkins DA
      Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy.
      ]. In normal karyotype AML, high expression of osteopontin was associated with a poor prognosis [
      • Powell JA
      • Thomas D
      • Barry EF
      • et al.
      Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML.
      ]. In addition, it has been reported that matrix stiffness differentially regulates myeloid leukemias, as well as their sensitivity to chemotherapeutic agents in vitro and in vivo [
      • Shin JW
      • Mooney DJ.
      Extracellular matrix stiffness causes systematic variations in proliferation and chemosensitivity in myeloid leukemias.
      ].
      In line with this, the interaction between collagen type I, but not fibronectin, and α2β1 integrin protects leukemia cells from doxorubicin-induced apoptosis in T-ALL cells [
      • Naci D
      • El Azreq MA
      • Chetoui N
      • et al.
      α2β1 integrin promotes chemoresistance against doxorubicin in cancer cells through extracellular signal-regulated kinase (ERK).
      ], as well as AML cells. In AML, this apoptosis was associated with activation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which was, however, inhibited by collagen [
      • Naci D
      • Berrazouane S
      • Barabe F
      • Aoudjit F
      Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation.
      ]. These data suggest that targeting integrins and, thereby, their interactions with the ECM may reduce chemoresistance in leukemias (Figure 3). The benefit of targeting E-selectin [
      • Barbier V
      • Erbani J
      • Fiveash C
      • et al.
      Endothelial E-selectin inhibition improves acute myeloid leukaemia therapy by disrupting vascular niche-mediated chemoresistance.
      ,
      • Godavarthy PS
      • Kumar R
      • Herkt SC
      • et al.
      The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin–SCL/TAL1–CD44 axis.
      ], as well as CD44, which binds hyaluronan, osteopontin, and E-selectin, in AML [
      • Jin L
      • Hope KJ
      • Zhai Q
      • Smadja-Joffe F
      • Dick JE
      Targeting of CD44 eradicates human acute myeloid leukemic stem cells.
      ] and CML [
      • Krause DS
      • Lazarides K
      • von Andrian UH
      • Van Etten RA
      Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells.
      ], has been studied.
      Figure 3
      Figure 3Leukemia and the extracellular matrix. Targeting the extracellular matrix as an innovative adjunct strategy for the treatment of leukemias, as detailed in the text. MMP-9=matrix metallopeptidase-9; CCL2=C–C motif ligand 2; MRD=minimal residual disease; TGF-β1=transforming growth factor β1; PTH=parathyroid hormone; ILK=integrin-linked kinase.

      New technologies for understanding the ECM

      Extensive and exciting developments in the field of mechanotransduction, partially because of the availability of novel materials for two-dimensional patterning of the ECM or three-dimensional scaffolds with nanoscale dimensions, hydrogels providing a gradient of soluble factors, ECM microarrays [
      • Gattazzo F
      • Urciuolo A
      • Bonaldo P
      Extracellular matrix: a dynamic microenvironment for stem cell niche.
      ], molecular tension-sensing probes, which are based on fluorescence resonance energy transfer (FRET), high-resolution time-lapse imaging and genetic labeling approaches have facilitated the identification of biophysical properties of the ECM.
      Atomic force microscopy (AFM), for example, allows multifunctional imaging and manipulation of biological samples on a single molecule to a living cell level [
      • Dufrene YF
      • Ando T
      • Garcia R
      • et al.
      Imaging modes of atomic force microscopy for application in molecular and cell biology.
      ] and simultaneous mapping of mechanical and kinetic properties of ligand–receptor bonds [
      • Alsteens D
      • Pfreundschuh M
      • Zhang C
      • et al.
      Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape.
      ]. AFM can measure changes in the force-dependent lifetime of single bonds between FN and α5β1 integrin in the piconewton range at steady state as well as after chemical activation [
      • Kong F
      • Garcia AJ
      • Mould AP
      • Humphries MJ
      • Zhu C
      Demonstration of catch bonds between an integrin and its ligand.
      ,
      • Kong F
      • Li Z
      • Parks WM
      • et al.
      Cyclic mechanical reinforcement of integrin–ligand interactions.
      ]. With high-resolution traction force microscopy, characterization of the distribution and dynamics of traction forces within single focal adhesions in fibroblasts in response to ECM stiffness has become possible [
      • Plotnikov SV
      • Pasapera AM
      • Sabass B
      • Waterman CM
      Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration.
      ]. FRET technology has been reported to quantify the number of bonds between oligopeptides and cellular receptors in a three-dimensional gel matrix, while simultaneously providing insight into cellular proliferation [
      • Kong HJ
      • Boontheekul T
      • Mooney DJ
      Quantifying the relation between adhesion ligand-receptor bond formation and cell phenotype.
      ] or substrate stiffness [
      • Kong HJ
      • Polte TR
      • Alsberg E
      • Mooney DJ
      FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.
      ]. The single-cell force spectroscopy technique also measures adhesive interactions of single living cells with the ECM and cells within seconds to minutes of the establishment of cell–ECM interactions [
      • Friedrichs J
      • Helenius J
      • Muller DJ
      Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy.
      ,
      • Taubenberger A
      • Cisneros DA
      • Friedrichs J
      • Puech PH
      • Muller DJ
      • Franz CM
      Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy.
      ].
      Genetic labeling approaches of ECM proteins such as fibronectin with a green fluorescent protein (GFP) tag allows observation of the stretching and tension by fluorescence [
      • Ohashi T
      • Kiehart DP
      • Erickson HP
      Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin–green fluorescent protein.
      ], as well as the dynamics of focal adhesions formed in migrating cells [
      • Laukaitis CM
      • Webb DJ
      • Donais K
      • Horwitz AF
      Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.
      ].
      In summary, excellent tools are now available to help us understand the impact of mechanical forces of the ECM in the BMM on the behavior of HSPCs and LSCs, which may lead to innovative therapies for regenerative medicine and hematological cancers.

      Conclusions

      Decades of research have been dedicated to understanding the HSC-intrinsic pathways governing HSC biology. However, there is a growing body of evidence indicating that the BMM provides necessary signals modulating HSC homeostasis. In addition to the contributions of cellular components of the BMM, many reports now suggest that the acellular ECM in the BMM also influences HSC and LSC functions such as adhesion, migration, maintenance of stemness, and differentiation via receptors on HSC- and LSC-binding proteins of the ECM. In turn, the generation, structure, and deposition of ECM proteins are modulated by leukemias, leading to altered secretion of cytokines, growth factors, and MMPs promoting leukemia progression. Given early evidence of the ECM's involvement in leukemia progression, a more thorough understanding of the role of the ECM in normal and malignant hematopoiesis is essential. This would likely pave the way for more efficient eradication of LSCs—the requisite for cure.

      Acknowledgments

      This work was supported by the Deutsche Kinderkrebsstiftung (Grant DKS 2016.16 to DSK) and the LOEWE Center for Cell and Gene Therapy Frankfurt (CGT) and by institutional funds of the Georg-Speyer-Haus to DSK. The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health (BMG) and the Ministry of Higher Education, Research and the Arts of the State of Hessen (HMWK). The LOEWE Center for Cell and Gene Therapy Frankfurt is funded by HMWK, Reference No. III L 4–518/17.004 (2010).

      Conflict of interest disclosure

      DSK holds Patent No. WO2018/046666 for the use of fibronectin and ILK inhibitors in leukemia and Patent WO2020016346A1 for the use of periostin for the treatment of hematological complications.

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