Targeted drug delivery for tumor therapy inside the bone marrow

Accepted Manuscript Targeted Drug Delivery for Tumor Therapy inside the Bone Marrow

Chao-Feng Mu, Jianliang Shen, Jing Liang, Hang-Sheng Zheng, Yang Xiong, Ying-Hui Wei, Fanzhu Li PII:

S0142-9612(17)30764-0

DOI:

10.1016/j.biomaterials.2017.11.029

Reference:

JBMT 18367

To appear in:

Biomaterials

Received Date:

29 June 2017

Revised Date:

26 October 2017

Accepted Date:

21 November 2017

Please cite this article as: Chao-Feng Mu, Jianliang Shen, Jing Liang, Hang-Sheng Zheng, Yang Xiong, Ying-Hui Wei, Fanzhu Li, Targeted Drug Delivery for Tumor Therapy inside the Bone Marrow, Biomaterials (2017), doi: 10.1016/j.biomaterials.2017.11.029

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ACCEPTED MANUSCRIPT Targeted Drug Delivery for Tumor Therapy inside the Bone Marrow Authors: Chao-Feng Mu1,*, Jianliang Shen2,3, Jing Liang4, Hang-Sheng Zheng1, Yang Xiong1, YingHui Wei1, Fanzhu Li1,* Affiliations: 1

Department of Pharmaceutics, College of Pharmacy, Zhejiang Chinese Medical University,

Hangzhou, Zhejiang 310053, China 2

Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences,

Wenzhou, Zhejiang 325001, China 3

School of Ophthalmology & Optometry, School of Biomedical Engineering, Wenzhou

Medical University, Wenzhou, Zhejiang 325035, China 4

Department of Biochemistry, Virginia Polytechnic Institute and State University,

Blacksburg, Virginia 24060, USA Corresponding Authors: * Department of Pharmaceutics, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China. Phone: +86 571 61768158; Fax: +86 571 61768136; Email address: [email protected] (C.F. Mu), [email protected] (F. Li) Notes The authors declare no competing financial interest.

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ACCEPTED MANUSCRIPT Graphic Abstract

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ACCEPTED MANUSCRIPT Abstract Bone marrow is the primary hematopoietic organ, which is involved in multiple malignant diseases including acute and chronic leukemia, multiple myeloma, myelodysplastic syndromes, and bone metastases from solid tumors. These malignancies affect normal homeostasis and reshape the bone marrow microenvironment. There are limited treatment options for them because of their inevitable aggravation. The current systemic administration of anticancer agents is difficult to achieve ideal therapeutic dose to suppress tumor growth at bone marrow diseased sites, and is always associated with a high incidence of relapse and severe side effects. The limitations of current treatments urge scientists to develop bone marrow targeted drug delivery systems intended for the treatment of diseased bone marrow, which can improve the efficacy of therapeutic agents and reduce their dose-limiting systemic side effects on healthy tissues. In this review we first present the current opinions on bone marrow vasculature, as well as the molecular and structural interactions between tumor cells and the diseased bone marrow. In the second part, we highlight the different design rationales and strategies of bone marrow delivery systems and their therapeutic applications for the treatment of malignancies inside the bone marrow.

Key words: bone marrow microenvironment, leukemia, bone metastasis, chemotherapy, molecular target therapy, drug resistance

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ACCEPTED MANUSCRIPT 1. Introduction Bone marrow is the primary hematopoietic organ located in the cavities of vascularized bones. It contains hematopoietic and mesenchymal stem cells. The hematopoietic stem cells (HSCs) differentiate into myeloid and lymphoid lineages of cells. The mesenchymal stem cells (MSCs) give rise to several different lineages, such as osteoblasts, fibroblasts, endothelial cells and other stromal cells [1]. All these types of cells, along with extracellular matrix (ECM) and microvessels comprise the bone marrow microenvironment in a spongelike texture. MSCs mainly support the hematopoiesis process via direct cell-cell contact or indirect interactions with HSCs or progenitor cells by releasing soluble factors (chemokines, cytokines and growth factors etc) into the bone marrow [2]. Multiple malignant diseases are related to the bone marrow including multiple myeloma, myelodysplastic syndromes, leukemia, and bone metastases originated from primary tumors. Moreover, these diseases, in turn, also affect normal homeostasis and reshape the bone marrow microenvironment [3]. Cytokines and growth factors secreted by tumor and other bone marrow cells are regulated through autocrine and paracrine signaling pathways within the bone marrow microenvironment. Aberrant variations in the osteoblasts, fibroblasts, endothelial cells, ECM components, cytokines, and growth factors adapt the survival and growth of these malignant tumor cells. Bone marrow angiogenesis also elevates in hematologic malignancies like acute myeloid leukemia, chronic lymphatic leukemia, non-Hodgkin lymphomas, and multiple myeloma etc [4]. Bone metastasis and multiple myeloma can lead to the enhanced activity of osteoclast cells resulting in pain, hypercalcemia, spinal cord compression and pathological fracture in their late stages [5, 6]. There are no curative treatment approach for tumors within the bone because of the inevitable aggravation of blood cancers and bone metastatic solid tumors in clinic [7]. It is difficult to achieve enough therapeutic dose of anticancer agents at tumor sites inside the bone marrow to suppress the tumor growth via intravenous administration [8]. Chemotherapeutic agents are always administered in high doses or frequently in order to reach the effective therapeutic magnitude inside the diseased bone marrow, which may lead to severe side effects including myelosuppression and dose-limiting toxicity to healthy tissues. On the other hand, bone marrow microenvironment induces resistance of tumor 4

ACCEPTED MANUSCRIPT cells, especially cancer initiating/stem cells to chemotherapy and makes them have the proliferation potential to mediate disease relapse. Therefore, targeting tumors existing in the bone marrow remains a huge challenge owing to the low drug availability in marrow and microenvironment caused drug resistance [7]. All these limitations of current bone marrow residing tumor treatments urge us to develop bone marrow targeted delivery carriers of therapeutic agents intended for improvement of the therapeutic efficacy and minimize the dose-limiting toxicities on healthy tissues. Bone marrow targeted delivery strategies may provide great potential for precise diagnostics, and/or therapeutics of malignancies within the bone marrow [9]. 2. Bone marrow structure 2.1 Vasculature in the bone marrow There are huge numbers of blood vessels and capillaries traversing through the bone and bone marrow. The vasculature of bone marrow is comprised of a functional and complex microvessel network that offers nutrients and oxygen, structure support, and egress for the mature blood cells [10]. Moreover, the bone and bone marrow microvessel network are extensively interconnected. Intraosseous blood vessels, which exist unevenly inside the trabecular or cortical bone, connect with marrow vessels and run continuously within the interior of bone (Figure 1A) [11]. Meanwhile, the microvessels distribute randomly in the bone (Figure 1B), and the blood flow move through the winding paths and change the speed through bone and bone marrow. The sluggish blood flow facilitates the blood cells to interact with endothelial cells via cell adhesion molecules in the sinusoids [11]. The endosteum is a thin connective layer that exists on the interface of bone and bone marrow [12]. There is an abundance of arterioles and sinusoids close to the endosteum (Figure 1C). The nascent bone marrow cavity excavated by osteoclasts contributes to the transformation of arterioles into large sinusoids (Figure 1D).

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Figure 1 Vasculature in the bone and bone marrow. (A) The distribution of blood vessels in the trabecular and cortical bone. Adapted with permission from [12]. (B) The intracortical vessel network opening in the bone marrow at the bone surface (white arrows) in the microradiograph of a rat tibia section (50 μm). The black arrow indicates the vessel looping in the bone and reentering the bone marrow. Adapted with permission from [11]. (C) The vessel network (red) from the bone marrow towards the endosteal surface (blue) in the three-dimensional reconstructed microphotograph from 50 μm below the surface. Adapted with permission from [12]. (D) The cross-section of blood vessels along the endosteal surface (blue). EV, endosteal vessels; S, sinusoid; white arrow, the transition from arterioles to sinusoidal vessels. Adapted with permission from [13]. The blood vessels between the bone marrow and the peripheral circulation form an extremely thin functional barrier (mostly less than 2-3 μm), termed as the marrow-blood barrier. It is comprised of sinuses with a continuous endothelial cell layer and an uncontinuous adventitial reticular cell layer [14]. Sinusoids are specialized venules that constitute a reticular structure of fenestrated capillaries which permit cells to ingress and 6

ACCEPTED MANUSCRIPT egress of the circulation [15]. There are relatively large fenestrations (30-40 μm in diameter) in the endothelium of sinusoidal capillaries. These sinusoidal capillaries allow mature blood cells (7.5-25 μm), serum proteins and foreign particulates to pass and this process is facilitated by a basal lamina, which is triggered via the interaction between these constituents and the endothelium [16]. On the other hand, it was reported that tumor cells migrating into bone marrow first adhered to the sinusoidal endothelium and then negotiated with the marrow-blood barrier. Noteworthy, tumor cells that transmigrate through the barrier successfully attribute to their selective binding to the adventitial reticular cell network and the extra sinusoidal matrix [14]. Blood vessels are required to deliver vital nutrients and oxygen for tumor progression [17]. Endothelial cells with functional and morphological features have the ability to induce neovascularization, promote tumor growth via paracrine growth factors and signals [18]. Angiogenesis plays a key role not only in solid tumors but also in hematologic malignancies like acute and chronic leukemia, lymphoma, myelodysplastic syndromes, and multiple myeloma [19]. A significantly higher bone marrow microvascular density along with increased serum vascular endothelial growth factor (VEGF) levels was observed in patients with hematologic malignancies compared with healthy volunteers [20]. The angiogenesis level in the bone marrow of patients with different types of leukemia is associated with their therapeutic implications and prognosis [4, 21]. 2.2 Stem cell niches The stem cell niche is a physical and functional entity, which is essential and supportive for the long-term self-renewal and differentiation of stem cells [11]. Stem cells are located at the endosteum in close proximity to osteoblasts (endosteal niche) and also associated with vascular endothelial cells around sinusoidal vessels (vascular niche) in the bone marrow cavity (Figure 2). It has been defined by the association of specific stromal cells and surroundings with secretion of particular soluble signaling molecules [3]. It consists of a cellular compartment (osteoblasts, osteoclasts, endothelial cells and other stromal cells) and a noncellular compartment including the ECM and soluble factors [22]. Unlike any other fixed niches, the stem cell niche is transient and migratory [11]. It plays an important role in the survival, differentiation, proliferation, migration, and drug resistance of malignant tumor cells 7

ACCEPTED MANUSCRIPT inside the bone marrow.

Figure 2 The endosteal and vascular niches inside the bone marrow. The endosteal and vascular cells contribute to the formation of HSC niches. (A) Schematic illustration of endosteal and vascular niches. Osteoblasts, osteoclasts and other stromal cells produce various factors involved in HSC regulation. The endothelial cells, perivascular reticular cells and mesenchymal stromal cells participate in regulating HSCs around sinusoids. (B) A typical vascular niche complex with HSCs in the bone marrow (*, sinusoid lumens). HSCs (white arrow) are labeled as CD150+CD48–CD41–lineage– (Red: CD150; green: CD48, CD41 and lineage). Adapted with permission from [23]. The regular stem cell niche as the new host microenvironment is not well adapted to the tumor cells that differentiate and metastasize into it. Notably, the niche can be altered by the leukemic or solid tumor cells, resulting in bone marrow substitution with leukemia or metastasis. Kaplan et al demonstrated that bone marrow-derived hematopoietic progenitor cells expressing vascular endothelial growth factor receptor 1 (VEGFR1) and very late antigen 4 (VLA-4) in bone marrow niches initiate the pre-metastatic site for tumor cell engraftment [24]. The transformed aberrant stem cell niches further drive tumor cell survival, proliferation and regulate chemotherapy resistance. 2.3 Interactions between the bone marrow and endosteum 8

ACCEPTED MANUSCRIPT The endosteum as the interface of bone and bone marrow is covered with bone-forming osteoblasts, bone-resorbing osteoclasts and bone-lining cells. All these cells are derived from specific progenitor cells in the bone marrow. Osteoblasts and bone-lining cells are derived from MSC, a common progenitor cell with adipocytes [25]. Osteoclasts are differentiated multinucleated cells originated from mononuclear cells of HSC precursors along the myeloid lineage [26]. A great number of internal and external cues manipulate the delicate balances of the differentiation of MSC and HSC precursors. These different factors induce various signaling pathways and activate multiple transcription factors that facilitate these precursors to commit to osteoblast- or osteoclast-lineage in the bone marrow. In abnormal pathologic conditions such as multiple myeloma and bone metastases from solid tumors, the tumor cells produce chemical mediators including Interleukin 6 (IL-6) and receptor activator of nuclear factor κ-B ligand (RANKL) to stimulate the activity of osteoclasts in the bone marrow [22, 27]. The increase in osteoclast formation and activity lead to bone resorption exceeding formation which results in painful osteolytic lesions and increased bone fracture risks. On the other hand, Osteoblasts and osteoclasts directly regulate the HSC endosteal niche. Although the leukemic cells do not cause the imbalance of bone remodeling between bone formation and resorption, they alter their neighboring microenvironment near the endosteum. The isoforms of CD44 bind to hyaluronic acid expressed around the endosteum, which is able to help leukemia stem cells (LSCs) to inhabit in the bone marrow for leukemia progression [28]. 3. Malignancies inside the Bone Marrow There are a lot of abnormal conditions and diseases including the malignancies or infections able to affect the function and reshape the structure of the bone marrow. Correspondingly, the bone marrow microenvironment also promotes the disease progression and influences their response to therapeutic agents inside bone marrow. In this part, the most typical malignancies involved in bone marrow are reviewed. The molecular interactions between tumor cells and the bone marrow microenvironment, as well as the current therapeutic situations of these malignancies are highlighted. 3.1 Multiple myeloma Multiple myeloma is an aggressive hematologic disease caused by the accumulation and 9

ACCEPTED MANUSCRIPT proliferation of neoplastic plasma cells in the bone marrow [29]. It accounts for approximately 10% of hematologic cancers [30]. Multiple myeloma cells migrate into the bone marrow and disseminate in specialized niches facilitating their long-term survival [31]. SDF-1/C-X-C chemokine receptor-4 (CXCR4) is a critical regulator of multiple myeloma homing to bone marrow microenvironment and formation of neoangiogenic niches. This chemokine-receptor axis can be as a desirable target for therapeutics to abrogate multiple myeloma trafficking [32]. Meanwhile, osteoblasts may also maintain the survival and growth of multiple myeloma cells by secreting IL-6 [22]. Specifically, the interaction between multiple myeloma cells and stromal cells triggers nuclear factor κ-B (NF-κB) signaling pathway and IL-6 secretion in stromal cells [4, 33]. This paracrine loop alters the bone marrow milieu for multiple myeloma cell growth. Furthermore, multiple myeloma cells are also able to adhere to bone marrow stromal cells and ECM via adhesion proteins such as vascular cell adhesion molecule 1 (VCAM-1), type I collagen and fibronectin, which activates many pathways resulting in upregulation of cell cycle regulators and antiapoptotic proteins [34]. In the past decades, the median survival of patients with multiple myeloma was prolonged greatly, attributed to autologous stem cell transplantation and treatments with drug combinations (bortezomib, thalidomide, melphalan and cyclophosphamide etc) [35]. In addition, bisphosphonates (pamidronate and zoledronic acid) are also administered to prevent pathological fracture caused by multiple myeloma. Although all these treatments do induce durable remission and prolong the overall survival, they are not curative to patients with multiple myeloma because of the failure to eradicate tumor cells inside the bone marrow. 3.2 Acute myeloid leukemia Acute myeloid leukemia (AML) is a malignant disease characterized by high proliferation of abnormal white blood cells with consequent interference of normal hematopoiesis in the bone marrow [36]. AML treatment options highly lie on its specific subtype and other conditions of patients. Chemotherapy as the primary choice for AML treatment, the response of patients to single chemotherapeutic agent is short lived and not curative. Combination of two or three anticancer drugs including cytarabine, 6-thioguanine, idarubicin, hydroxyurea 10

ACCEPTED MANUSCRIPT and mitoxantrone etc are often used for AML therapy [37, 38]. Chemotherapy for AML always has various side effects depending on the drugs used [39]. Furthermore, comprehensive mutational profiling among the diverse subtype of AML patients will enable consideration of molecular target therapy, either alone or in combination with chemotherapeutics, which will improve the overall treatment outcomes [40]. Additionally, AML as a rapid progressive cancer is always characterized by an obvious augment of microvessel density in the bone marrow [41]. AML patients showed increased levels of AML blasts associated VEGF and neovascularization compared to normal bone marrow [42]. Thus, VEGF targeted agents also can be used as novel anti-angiogenic therapies in AML [43]. AML as a cell autonomous disorder, its genetic mutant events lead to the conversion of normal hematopoietic cells for the generation of leukemia. Due to the functional heterogeneity of AML cells, a small population of LSCs has long-term differentiation potential and the capability to proliferate and eventually retain the AML subtype [44]. The LSCs are generally quiescent and close to the osteoblast-rich endosteal region within the bone marrow microenvironment. The interactions between AML blasts and bone marrow microenvironment mediated by stroma-secreting soluble factors and cell-cell contact are involved in chemotherapy and molecular therapy resistance in AML [45]. 3.3 Chronic myeloid leukemia Chronic myeloid leukemia (CML) as a myeloproliferative disorder originates from a primitive hematopoietic cell transformed by the BCR-ABL oncogene, which defines the subsequent molecular events of leukemia [46]. The deregulated BCR-ABL tyrosine kinase activity is responsible for modulating different signaling pathways, and consequently leads to increased hematopoietic stem and progenitor cell proliferation. It also disturbs the interaction of hematopoietic stem and progenitor cells with the ECM and stroma, causing abnormal expansion of differentiated myeloid cells [47]. The constitutive BCR-ABL tyrosine kinase activity can be effectively inhibited with small molecule tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, dasatinib and ponatinib etc [48]. TKI therapy targeting the BCR-ABL oncogene, has transformed CML to a non-life threatening disease. However, recent findings suggest that most patients with CML usually 11

ACCEPTED MANUSCRIPT relapse after discontinuation of TKI treatment due to the existence of residual leukemia cells in the bone marrow [49]. Primitive CML progenitors are not oncogene addicted, and a small population of LSCs in the bone marrow is likely the source of disease recurrence after TKI cessation [50]. Evidence indicates that the bone marrow microenvironment (stromal cells or ECM) protects primitive CML cells/LSCs from the effects of chemotherapy and small molecule targeted therapy by providing critical BCR-ABL-independent survival cues to cells [51-53]. Most studies suggest that the eradication of LSCs may require either targeting their self-renewal mechanisms or blocking interactions with the bone marrow microenvironment [49]. On the other hand, vascular adverse events (VAEs) have emerged as potential side events with TKI therapy in CML patients [54]. The main VAEs identified so far are peripheral arterial occlusive disease, which mainly occurs in patients long-term receiving second or third-generation TKIs treatment [55]. There is also some evidence that the frequency of VAEs is triggered in a dose-dependent manner [56]. The rates of severe VAEs can be decreased greatly by lowering the dose or shortening the exposure time via altering the administration route of TKIs and increasing their bioavailability in vivo [57]. 3.4 Bone metastasis Bone is one of the most common organs for solid tumor metastasis in advanced cancer patients [58]. The close interactions between tumor cells and the bone marrow microenvironment facilitate the seeding and growth of metastatic tumor cells in the bone. Tumor cells that circulate in the blood invade and negotiate with sinusoids in the bone marrow to initiate the bone metastasis process. During bone metastasis, the interaction between tumor cells and marrow microenvironment perturbs the normal bone metabolism and promotes the formation of bone metastatic foci (Figure 3). Osteoblastic cells participate in the tumor cell colonization and control their dormancy in the bone marrow [59]. Subsequently, tumor cells stimulate the secretion of cytokines to elevate the osteoclasts activity via interleukins (ILs), the parathyroid hormone-related protein (PTHrP), and transforming growth factor β (TGFβ) etc [7]. The serial changes in the microenvironment increase the bone resorption and provide access to the demineralized bone surface.

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Figure 3 Bone metastasis from solid tumors. (A) The interactions of solid tumor cells with bone marrow constituents, resident osteoblasts and osteoclasts in bone metastasis. (B) The bone metastasis process of human breast tumor in athymic nude mice monitored by bioluminescence after intracardiac injection of MDA-MB-231 cells. (C) H&E-stained section of MDA-MB-231 tumor nodule inside the upper trabecular bone of mouse femur. The interface between bone marrow cells and dense tumor cells is clear (dotted line). BMPs, bone

morphogenetic

protein;

FGFs,

fibroblast

growth

factors;

IGFs,

insulin-

like growth factors. Patients with highly disseminated bone metastases are usually inoperable. Although each step in the metastatic process represents a potential target for treatment to abrogate the bone metastasis, it is still not curative due to the poor bone distribution of intravenously injected anticancer agents and chemotherapy resistance of tumor cells packed with the bone marrow [7, 8]. Here, we mainly emphasize the IL-6-caused chemotherapy resistance in 13

ACCEPTED MANUSCRIPT bone metastasis treatment. Tumor cells stimulate IL-6 secretion in bone marrow mononuclear cells inside the metastatic bone marrow. Similarly, tumor cells from prostate and breast cancer also secrete high amounts of IL-6 autocrinely [27]. IL-6 inflammatory feedback loop results in the self-renewal of cancer stem-like cells and induction of epithelial to mesenchymal transition, both of which are implicated in therapeutic resistance [60]. IL6/Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling promotes tumor development through the stromal surroundings in solid and metastatic tumor niche [61, 62]. The tumorigenicity of HR−/HER2+ breast cancers was restrained via inhibiting the activity of IL-6/JAK2/STAT3-calprotectin signaling pathway [63]. Thus, combination of JAK2 inhibitors and chemotherapeutic agents may have the advantage of overcoming chemotherapy resistance caused by bone marrow microenvironment for bone metastasis treatment. 4. Bone marrow drug delivery strategy

Figure 4 Schematic illustration of bone marrow targeted drug delivery strategies. The drug availability is often quite low when therapeutic agents are administrated systematically for the treatment of malignancies in the bone marrow such as leukemia and bone metastases due to their rapid metabolism and clearance, sometimes even before they are capable to affect their targeted diseased sites in the bone marrow. Most of the injected drugs are either cleared due to the body’s metabolic/excretory system, or accumulate in 14

ACCEPTED MANUSCRIPT other highly perfused tissues/organs prior to reaching the bone marrow. Therefore, drugs are always administered in high doses and/or frequently for reaching the effective therapeutic magnitude inside the bone marrow, which can lead to inevitable systemic side effects [64]. To overcome this challenge, the design rationale demands careful consideration of the drug conjugates/carriers and especially ensuring that the drug selectively reaches the lesion or tumor site inside the bone marrow [65]. Currently, there are mainly five different drug delivery strategies applied to bone marrow residing tumor therapy (Figure 4). Summary of rationales and applications of bone marrow-targeted delivery systems is presented in Table 1. Table 1 Rationales and applications of bone marrow-targeted delivery systems Delivery carrier

Targeting rationale

Drug

Application

Ref

PLGA nanoparticle

passive targeting via its neutral charge and small size (about 150 nm) SA increases liposome capture by macrophage in BM anionic low-cholesterol liposome promotes accumulation in BM (AspSerSer)6 binds to calcium phosphate in bone forming surface

paclitaxel

[8]

PEG-PLGA nanoparticle

alendronate-HA binding in bone matrix

bortezomib

PEG-PLA micelle

alendronate-HA binding in bone matrix

ponatinib, SAR302503

PLGA nanoparticle

Poly aspartic acid binds to HA

----

HPMA polymer conjugate

alendronate-HA binding in bone matrix

paclitaxel

immunocytokine

F8, F16 antibody recognize fibronectin and tenascin-C at AML

interleukin 2

slow bone metastasis and inhibit bone loss in prostate cancer bone metastatic mice 99mTC labeling imaging in rabbits and monkeys indicates its BM-targeted efficiency increase drug accumulation in leukemia cells inside BM and enhance efficacy in AML mice deplete Plekho1 in osteogenic cells and increase the bone mass in both healthy and osteoporotic rats pretreatment inhibits myeloma growth better than free drug in myeloma mice model extend survival time of BaF3/T315I cells inoculated CML mice specific binding to the bone tissue without cytotoxicity to osteoblasts inhibit breast tumor growth in 4T1 cell intra-tibia injected mice model promote complete tumor eradication in combination with cytarabine in AML

liposome

liposome

liposome

----

cytarabine: daunorubicin Plekho1 siRNA

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[66, 67] [68, 69] [70]

[7]

[57]

[64]

[71]

[72]

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porous silicon particle

angiogenesis site in BM E-selectin thioaptamer binds to E-selectin

porous silicon particle

E-selectin specific thioaptamer

parthenolide

liposome

folate binds to FR-β

doxorubicin

nanostructured lipid carrier

dual targeting via alendronate-HA and folate-FR-β bindings

mitoxantrone

99mTC,

STAT3 siRNA

xenograft mice model extend survival time of bone metastatic mice by inhibition of STAT3 expression in tumor impairment of LSCs in AML secondary transplantation mice folate-modified liposomes increase median survival times of AML mice deliver drugs to LSCs in bone marrow for refractory leukemia therapy

[73]

[74]

[75]

[76]

Technetium-99m; BM, bone marrow; HA, hydroxyapatite; FR-β, folate receptor-β

4.1 Passive targeting Passive targeting refers to the accumulation of drug-containing carriers or drugconjugates at a specific disease site through leaky vasculatures including tumors, infarcts, inflammation etc that can avoid the elimination due to body defense mechanisms [77]. The size and surface properties of drug carriers must be controlled specifically to avoid uptake by the mononuclear phagocyte system (MPS) to maximize circulation time and targeting disease ability. The biodistribution of particulates largely depends on their properties such as size, surface charge, shape, and other surface characteristics [78]. The liver, spleen and bone marrow have capillaries with large pores or openings under normal circumstances. For bone marrow drug delivery, the drug accumulation amounts in the bone are ascribed to the uptake of particulates by phagocytic reticulo-endothelial cells of sinusoid vessels in the bone marrow [79]. The endothelium of vascular sinusoids collects particulates from the blood circulation via both transcellular and intercellular routes. The transcellular route takes place through the fenestrae between sinusoidal endothelial cells, which highly depends on the particle size [9]. The sinusoidal capillaries have relatively large openings (30-40 μm) which allow mature blood cells (7.5-25 μm) and various serum proteins to pass. However, Moghimi et al reported that the size of the fenestrae in endothelial wall is only between 85-150 nm. It becomes more difficult to pass through the sinusoids when the particle size is above 150 nm [16]. It can be speculated that the fenestrae are dynamic as well as their numbers and diameter readily varies. They can completely disappear or appear 16

ACCEPTED MANUSCRIPT in large numbers, which depends on the negotiation between particulates and the sinusoidal endothelium [80]. On the other hand, for improving drug delivery efficiency into bone marrow, intravenously injected particulates firstly need to circulate in the blood long enough to pass through the sinusoidal capillaries. To increase the blood circulation time, modification of poly(ethylene glycol) onto the surface of particulates can lower their uptake by macrophages in the liver and spleen. Furthermore, neutral PLGA nanoparticles have the relatively low possibility of being opsonized by the organs of the MPS compared with anionic and cationic PLGA nanoparticles. Therefore, neutral nanoparticles are more effective to accumulate in the bone marrow. The neutral paclitaxel-containing PLGA nanoparticles (around 150 nm) slowed down the development of prostate tumor bone metastasis and inhibited bone loss in intraosseous prostate cancer murine model with reduced side effects compared with its Cremophor EL formulation [8]. 4.2 Macrophage capture mediated bone marrow targeting Macrophages are distributed in nearly all tissues and are produced when circulating monocytes migrate and differentiate in specific tissue. Kupffer cells and splenic macrophages are responsible for the uptake of circulating vesicles in the MPS organs [81]. However, macrophages with access to the bone marrow as part of the MPS get little attention in comparison with the liver and spleen. Indeed, it is a potential target for bone marrow-selective drug delivery using nanocarriers. Macrophages express several kinds of surface receptors for the phagocytosis of specific surface modified particulates including liposomes and polymeric nanoparticles etc. The receptors available to target macrophages with nanocarriers are mannose receptor, galactose receptor, scavenger receptor, and nicotinic acetylcholine receptor etc [82-84]. However, it is tough to target macrophages in the bone marrow because these receptors are commonly expressed on macrophages in almost all tissues.

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Figure 5 Macrophage capture mediated bone marrow-targeted liposomes. (A) The lipid constituents of liposomal formulation. SA, L-glutamic acid-, N-(3-carboxy-1-oxopropyl)-,1,5dihexadecyl ester. Adapted with permission from [9]. (B-E) Distribution of Technetium-99mlabeling 10% SA-modified liposomes (SA-Ve) and control liposomes (Ve) containing different amounts of PEG-DSPE in the bone marrow (B), liver (C), spleen (D), and blood (E) at 24 hours after intravenous injection in rabbits (*, p < 0.01; †, p< 0.05). Adapted with permission from [66]. Recent studies demonstrated that some types of surface modified liposomes are able to accumulate into the bone marrow via specific phagocytosis [9]. It is speculated that this phenomena is resulted from the lipids delivered by macrophages for energy supply and membrane biosynthesis in the bone marrow. Sou et al found that liposomes surfacemodified with 10% of an anionic l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) were specifically distributed into bone marrow via selective uptake by rabbit bone marrow macrophages. By contrast, the distribution of liposomes in the liver and spleen 18

ACCEPTED MANUSCRIPT decreased greatly after SA modification (Figure 5) [66]. Low cholesterol

liposomal

formulation containing 20% 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) developed by Tardi et al achieved high level of bone marrow accumulation which is largely attributed to that the anionic charge of liposomes have relatively high affinity with the scavenger receptor on bone marrow macrophages [69]. Notably, the bone marrow targeting capability of surface-modified liposomes is dose-dependent. The lipid uptake by bone marrow macrophages would be saturated at high injected lipid dose, which can eliminate the organ selectivity [9]. Furthermore, most of injected liposomes are sufficiently uptake by macrophages and early osteoclastic precursors instead of mature osteoclasts in the bone marrow [85]. These findings support that some strongly negative charged liposomes containing anionic lipid derivatives have the potential to be used as bone marrow targeted nanocarriers via macrophage capture. Nonetheless, the exact selective mechanism of liposomes captured by bone marrow macrophage is still unclear. Elucidation of the mechanism will offer rationale for designing novel nanocarriers in the same targeting manner. In addition, liposomes as drug carriers are able to encapsulate small molecular, macromolecular, or multiple therapeutic agents with tunable surface properties and biocompatibility. Currently, scale-up production of liposomes with good reproducibility and process control has already been realized [86], which will greatly increase the translational potential of macrophage capture mediated bone marrow targeting strategy. 4.3 Bone surface binding-mediated bone marrow targeting Bone is comprised of a complex collagen matrix, non-collagenous proteins and hydroxyapatite (HA) crystals. Bone sialoprotein (BSP) and osteopontin (OPN) as the extracellular phosphoproteins are involved in the elaborate regulation of osteoclast binding and bone mineralization. There are plentiful glutamic acid or aspartic acid sequences in these extracellular phosphoproteins, which are responsible for their high affinity with HA crystals [87-89]. Several oligopeptides such as eight repetitive aspartic acids (Asp)8 have been demonstrated their particular interaction with bone tissues [90, 91]. The high affinity between oligopeptides and HA may attribute to the ionic interaction between the negativecharge of short peptides and the positive-charged calcium ion within HA crystals at 19

ACCEPTED MANUSCRIPT physiological pH. It was reported that (Asp)8 preferred binding to bone-resorption surfaces. The bone-resorption surfaces predominantly occupied with osteoclasts are constituted of highly crystallized HA [92]. It was demonstrated that (Asp)8 mainly binds to highly crystallized HA instead of low crystallized HA in vitro. By contrast, another type of oligopeptides-(AspSerSer)6 preferentially binds to the mantle dentin, which is composed of small amorphous calcium phosphate crystals instead of the well-oriented HA [93]. A targeting 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)-based cationic liposome attached with (AspSerSer)6 was designed for delivering an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (Plekho1) specifically to bone-formation surfaces. The Plekho1 siRNA was selectively enriched in osteogenic cells for subsequently depleting Plekho1 in rats. The Plekho1 siRNA loaded liposomes greatly promoted the bone formation and improved the bone micro-architecture in healthy and osteoporotic rats [70].

Figure 6 Bone microenvironment targeted alendronate-PEG-PLGA nanoparticles (Ald-NPs) loaded with bortezomib (Bort) for multiple myeloma therapy. (A) Schematic illustration of the interaction between Ald-NPs and bone matrix. (B) Fluorescence images of whole mouse body injected with Alexa647-labeled NPs by IVIS (acquired 24 hours post i.p. injection). (C) Total fluorescence intensity analysis of images from B. (D) The bioluminescence imaging signals from mice treated with bortezomib loaded Ald-NPs (Ald-Bort-NPs) were remarkably lower compared with that of empty Ald-NPs or free drug groups at the day of imaging. The mice were pretreated with empty Ald-NPs, free drug, or Ald-Bort-NPs for three weeks and 20

ACCEPTED MANUSCRIPT then inoculated with GFP+Luc+ MM1S cells. Adapted with permission from [7]. Bisphosphonates as another type of stable bone-targeting molecules display an exclusively high affinity with HA. Compared to bone-targeting oligopeptide-moieties such as (Asp)8 and (AspSerSer)6, bisphosphonates bind to both bone-formation and bone-resorption surfaces [92]. The exceptional characteristics of bisphosphonates make it a high efficient bone targeting moiety for delivery of antineoplastic compounds, radionucleotides into the bone marrow [76]. It’s worth noting that the binding of polymers/nanoparticles to the bone sections ex vivo does not reflect their interactions with bone surfaces in vivo. Several different factors determine the bone targeting efficiency of polymers/nanoparticles in the systemic circulation, including their surface properties and aqueous stability, density of bisphosphonates on the surface, the disposition by MPS, and the accessibility of bone surface [64]. Wang et al demonstrated the bovine serum albumin nanoparticles coated with PEI-PEG-thiol-bisphosphonates lacked bone targeting after systemic administration in vivo, despite their high affinity to HA in vitro [94]. Alendronate as one of the typical bone-targeting bisphosphates, was used as bone-targeting moiety to conjugate with water-soluble N-(2hydroxypropyl)-methacrylamide (HPMA) copolymer. The alendronate-HPMAs exclusively accumulated in the bone tissue. Low alendronate content per HPMA copolymer (1.5 mol%) sufficed for the good bone deposition efficacy. The molecular weight determined the circulation time and biodistribution of the HPMA copolymer conjugates. The alendronateHPMA with molecular weight at 50-100 kDa was suitable as drug carriers for bone-targeting delivery [95, 96]. Swami et al developed alendronate-conjugated PEG-PLGA nanoparticles encapsulating bortezomib for multiple myeloma therapy (Figure 6A). The optimized nanoparticles with 20% alendronate surface modification had the effective HA binding in vitro and markedly increased the distribution of nanoparticles inside bone tissue in vivo (Figure 6 B and C). The bortezomib loaded alendronate-nanoparticles had the ability to alter the bone marrow microenvironment to prevent myeloma development in mice burdened with multiple myeloma (Figure 6D) [7]. Alendronate-PEG-PLA micelles were employed as active bone targeting nanocarriers for ponatinib and SAR302503 entrapment. The bone targeting effect was achieved when the alendronate ratio on the surface of micelles increased to 40%. 21

ACCEPTED MANUSCRIPT Small amount of alendronate modification (less than 10%) on the surface of micelles did not increase their accumulation in bone tissue [57]. Notably, some studies have reported that bisphosphonates can slow the development of bone metastasis and reduce the risk of skeleton-related events by preventing osteoclast differentiation and bone resorption [97]. However, the survival time of bisphosphonate-treated patients was not extended compared with those treated with placebo in several clinical studies [5]. The HSCs are preferentially located in the cancellous bone. This location includes both the endosteum close to osteoblasts (endosteal niche) and sinusoidal vessels associated with other stromal cells (vascular niche) [98]. The osteoblasts which are responsible for bone formation, have a direct role in regulating HSCs function [99, 100]. The LSCs possess the approximate characteristics with HSCs, but participate in the initiation and maintenance of leukemia

rather than regular hematopoiesis [28]. Therefore, bone-targeting molecules

such as bisphosphates and (AspSerSer)6 which are capable to bind to bone-formation surfaces may precisely deliver therapeutic agents to the endosteal stem cell niche and contribute to eradicate the LSCs in bone marrow microenvironment for refractory leukemia therapy. 4.4 Vascular targeting The bone marrow microvasculature is composed of capillaries and specialized thin-walled sinusoids [10]. Furthermore, angiogenesis as the process of new blood vessel formation, performs an essential role in both solid tumor metastasis and leukemia development [101]. A high degree of neovascularization which is always associated with poor prognosis, was discovered in the bone marrow of patients with AML, multiple myeloma, acute lymphatic leukemia, chronic lymphatic leukemia (CLL), or non-Hodgkin lymphomas compared with healthy volunteers, [17, 102, 103]. There are a vast spectrum of endothelial surface receptors highly expressed on the inner lining of normal or neoangiogenic blood vessels including VEGFRs, E-selectin, ανβ3 integrin, Aminopeptidase N/CD13, intercellular cell adhesion molecule (ICAM), and VCAM-1 etc. They are readily available as binding molecules for long-circulating drug carriers and utilized to assist drug targeting to the tumor vasculature [104-106]. Vascular targeting refers to taking advantage of the unique characteristics of tumor blood 22

ACCEPTED MANUSCRIPT vessels for the selective delivery of therapeutic agents such as chemotherapeutic drugs, cytokines, oligonucleotides, radionuclides and photosensitizers to tumor sites [72, 107-110]. Several angiogenesis receptors are selectively high expressed in some leukemia types and can be efficiently targeted using specific ligands in vivo [17, 111-113]. One strategy is to develop tumor-homing immunocytokines. Vascular targeting antibody-conjugated IL-2 exhibited a better antitumor activity compared to nontargeted IL-2 [72, 109]. The immunocytokines have been evaluated in phase 2 clinical trials for the treatment of patients with solid tumors and leukemia [114]. F8, F16 antibodies selectively bind to the extra-domain A and extra-domain B domains of fibronectin and A1 domain of tenascin-C, respectively, at AML angiogenesis site in the bone marrow. F8 and F16 antibodies stained a large fraction of AML

and

acute

lymphoblastic

leukemia

(ALL)

bone

marrow

biopsies

in

immunohistochemical analysis demonstrated the strong and selective antigen expression in acute leukemia [72]. The side effects of IL-2 conjugated immunocytokines are relatively low. These immunocytokines have displayed promising therapeutic efficacy and safety profiles in cancer patients [115]. Figure 7 Bone marrow vascular endothelium targeted porous silicon particles for bone metastatic breast tumor and acute myeloid leukemia (AML) therapy. (A) Drug containing nanoparticles (polyplexes or micelles) exist inside the nanopores of ESTA-MSV. (B) Scanning electron microscope images of MSV and ESTA-MSV (porous silicon particles, 1 μm in diameter and 400 nm in height). (C) Fluorescence images of ESTA-MSV distributed in the bone marrow (green, endothelial cells; blue, nuclei; red, Cy5-ESTA-MSVs). (D) The survival of mice bearing bone metastatic MDA-MB-231 tumor treated with ESTAMSV/STAT3 siRNA (n = 8-9), (B-D, adapted with permission from [73]). (E) Schematic illustration of the experimental design of AML secondary transplantation (PDX, patientderived xenografts). (F) Parthenolide loaded ESTA-MSV (MSV-PTL) impairs LSCs inside the bone marrow in vivo. The percentage of human leukemic cells in mouse bone marrow from secondary transplants in the different treatment arms. Each circle represents an individual mouse. Error bars represent S.E.M. (E & F, adapted with permission from [74]). E-selectin is a cell-adhesion molecule expressed on inflamed endothelial cells activated 23

ACCEPTED MANUSCRIPT by inflammatory cytokines, which always exists in the vasculature of inflammatory and tumor sites [116]. It aids the homing and adhesion of metastatic tumor cells to the endothelial cells inside the bone marrow [117-119]. An E-selectin specific thioaptamer (ESTA), that binds to E-selectin with high binding affinity and minimal cross-reactivity compared to the other selectins was discovered using a DNA thioaptamer combinatorial library [120]. E-selectin thioaptamer-conjugated porous silicon microparticles (1 µm in diameter and 400 nm in height, ESTA-MSVs) (Figure 7A and B) were developed as siRNA and parthenolide carriers for the treatment of bone metastatic breast cancer and AML, respectively [73, 74]. PEGPEI/STAT3-siRNA polyplexes were entrapped into nanopores (40 to 80 nm) of ESTA-MSVs via sonication. The ESTA-MSVs have shown specific bone marrow endothelium targeting efficacy in vivo. Red fluorescent-labeled ESTA-MSVs co-localized with the E-selectinpositive endothelial cells compared to untargeted Cy5-MSVs inside femurs after intravenous injection of ESTA-MSVs into metastatic breast cancer mice. Most ESTA-MSVs exited sinusoidal vessels and penetrated into the perivascular area via binding to E-selectin in the bone marrow (Figure 7C). Meanwhile, the ESTA-MSV/STAT3-siRNA treatment strikingly extended the survival in bone metastatic breast cancer mice (Figure 7D). LSCs exist within and are sheltered by the bone marrow niche [121]. The ESTA-MSVs was also utilized to load parthenolide-micelles for delivering drug into the bone marrow niche. Two doses of ESTA-MSV-parthenolide administrated 2 weeks apart were able to significantly reduce AML burden and impair LSC function in patient-derived AML murine model (Figure 7E and F) [74]. In bone marrow microenvironment, leukemic cells adhere to and interact with the neovasculature to protect them from chemotherapy [122]. Even though the therapeutic responses of antiangiogenic monotherapy are modest, it is found that antiangiogenesis in bone marrow can benefit leukemia therapy in clinical studies [43]. Dual/multiple drug loading approach can be used to entrap antigangiogenic and chemotherapeutic agents simultaneously. Vascular homing ligands decorated nanocarriers not only can direct antigangiogenic agent to neovasculature to suppress angiogenesis and destroy the interaction of leukemic cells with endothelial cells, but also can contribute the released chemotherapeutic drugs to inhibit leukemic cell proliferation near the neovasculature in the 24

ACCEPTED MANUSCRIPT bone marrow. Such a vascular targeting co-delivery strategy can maximize the combination therapeutic efficacy for the treatment of hematologic malignancies. 4.5 Specific tumor cell targeting inside the bone marrow The development and safe application of elaborate bone marrow-targeted therapy will depend on the capability to specific deliver therapeutic agents to leukemic or metastatic tumor cells and limit the capture by healthy bone marrow cells after systemic injection. Folate receptor-β (FR-β) was upregulated in AML cells after all-trans retinoic acid (ATRA) treatment. Meanwhile, FR-β expression was strikingly elevated in the bone marrow engrafted with FR-β (+) human AML cells in NOD/SCID mice [123]. FR-β-targeted liposomal doxorubicin combined with receptor induction using ATRA increased the median survival time of L1210JF/KG-1 AML burdened mice compared to nontargeted liposomal doxorubicin [75]. Multistep targeted delivery system mediated via alendronate and folate-targeting moieties could precisely target to the CML minimal residual disease region and LSCs located in bone marrow microenvironment for refractory CML therapy [76]. The increased delivery of both entrapped drugs (cytarabine and daunorubicin, 5:1 molar ratio) via low cholesterol phosphatidylglycerol (PG)-containing liposomes (CPX-351) to leukemic cells in the bone marrow displayed a selective capture process occurred between liposomes and leukemia cells. It was speculated that the highly negative-charged liposomes may increase their binding to the receptors (class-B scavenger or low-density lipoprotein receptors etc) overexpressed in leukemic cells from AML patients [124-126]. Recently, CPX-351 (Vyxeos) was approved for the treatment of two types of high-risk AML: newly diagnosed therapyrelated AML and AML with myelodysplasia-related changes by FDA [127]. Aptamers have been broadly used as ligands for targeted tumor therapy because of their high selectivity and affinity to the specific target molecules including proteins and peptides in tumor sites [128]. Several aptamers targeting leukemic cells have been developed through both the protein- and/or cell-based SELEX (systematic evolution of ligands by exponential enrichment) technologies for diagnosis and/or treatment of hematological malignancies including leukemia and lymphoma [129]. Sgc8 aptamers were screened for the specific molecular recognition of ALL cells [130]. NOX-A12 spiegelmer as one mirror image of aptamers specifically antagonize CXC chemokine ligand 12 (CXCL12)/SDF-1 for LSCs 25

ACCEPTED MANUSCRIPT migration into the bone marrow, which is in clinical trial for indications in non-Hodgkin’s lymphoma, CLL and multiple myeloma [131]. It specifically neutralizes CXCL12/SDF-1 and sensitizes CLL cells to bendamustine and fludarabine in bone marrow stromal cells cocultures [132]. Nucleolin as an abundant nucleolar protein, its expression is correlated with the cell proliferation level and always upregulated in actively dividing cells including AML and CLL/small lymphocytic lymphoma [133, 134]. AS1411 aptamer as nucleolin aptamer can bind to nucleolin and mediate AS1411-modified nanoparticles deliver entrapped therapeutic agents inside leukemic cells directly [129]. To eradication of tumors residing in the bone marrow, the most ideal approach is to selectively kill tumor cells and spare normal bone marrow cells. Therefore, it is crucial to identify specific tumor cell markers and selectively targeted destruction of tumor cells. T-cell immunoglobulin mucin-3 was shown expression in many kinds of AML LSCs but not in normal HSCs, which would be used as a promising cell surface marker to specifically target AML LSCs [135, 136]. Secondly, the high throughput screened tumor cell targeting ligands such as short synthetic peptides and aptamers can be conjugated to therapeutic drugs directly or attached onto the surface of nanocarriers containing therapeutic drugs. By means of the high affinity and specificity of targeting moieties, dual-ligand bone marrow targeting approach can be designed and used to increase the bone marrow homing ability of drug carriers and simultaneously enhance the entrapped drugs into specific leukemic or metastatic tumor cells inside the bone marrow. 5. Conclusions and future perspectives Targeted delivery of therapeutic agents plays a pivotal role in the effective and safe treatment of malignancies inside the bone marrow. Further elucidation of the correlation between the modification of drug carriers with ligands and bone marrow targeting efficiency in vivo will guide more broad and robust approaches to therapies. It will make carriers have the ability to overcome the multiple biological barriers en route to the bone marrow diseased sites. On the other hand, targeting malignancies inside the bone marrow is still a biological issue due to the cancer stem/initiating cell existence and the bone marrow microenvironment induced resistances. Combination therapy with two or more therapeutic agents acting on multiple biological targets has exhibited a promising potential in 26

ACCEPTED MANUSCRIPT overcoming therapeutic resistance. Particularly, disruption of the anchoring interactions between tumor cells and bone marrow microenvironment via multiple inhibitors may achieve great clinical responses to increase sensitization of tumor cells to chemotherapy [137]. The stable co-encapsulation of multiple drugs into a single bone marrow targeted drug carrier might alter the pharmacokinetics profiles of combined drugs to achieve the synergistic effect for tumor therapy inside the bone marrow in vivo [138, 139]. Besides, the accumulation of therapeutic agents inside the bone marrow could lead to cumulative toxicity to normal hematopoiesis. The development of elaborate bone marrow-targeted systems is essential for specific delivery of therapeutic agents to tumor cells and minimizing the capture by healthy bone marrow cells in the bone marrow. Overall, current findings are promising for the development of effective and safe bone marrow targeted formulations, which are expected to benefit the treatment of patients with bone marrow malignancies in the near future. Acknowledgements Authors acknowledge financial support from the National Natural Science Foundation of China (81703713, 81373982 and 81473434), Research Fund for the Doctoral Program of Higher Education of China (20123322120002), and internal support from Zhejiang Chinese Medical University. The authors would also like to thank Xuefeng Li for her assistance. References [1] J.A. Martinez-Agosto, H.K. Mikkola, V. Hartenstein, U. Banerjee, The hematopoietic stem cell and its niche: a comparative view, Genes Dev. 21 (2007) 3044-3060. [2] K. Le Blanc, D. Mougiakakos, Multipotent mesenchymal stromal cells and the innate immune system, Nat Rev Immunol. 12 (2012) 383-396. [3] A. Colmone, M. Amorim, A.L. Pontier, S. Wang, E. Jablonski, D.A. Sipkins, Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells, Science. 322 (2008) 1861-1865. [4] K. Podar, D. Chauhan, K.C. Anderson, Bone marrow microenvironment and the identification of new targets for myeloma therapy, Leukemia. 23 (2009) 10-24. [5] L.J. Suva, C. Washam, R.W. Nicholas, R.J. Griffin, Bone metastasis: mechanisms and therapeutic opportunities, Nat Rev Endocrinol. 7 (2011) 208-218. [6] I. Djunic, I. Elezovic, M. Marinkovic, N. Suvajdzic-Vukovic, D. Tomin, G. Jankovic, J. Bila, D. Antic, A. Vidovic, B. Neskovic, D. Nikolic-Vukosavljevic, Osteolytic lesions marker in multiple myeloma, Med Oncol. 28 (2011) 237-240. [7] A. Swami, M.R. Reagan, P. Basto, Y. Mishima, N. Kamaly, S. Glavey, S. Zhang, M. Moschetta, D. Seevaratnam, Y. Zhang, J. Liu, M. Memarzadeh, J. Wu, S. Manier, J. Shi, N. Bertrand, Z.N. Lu, K. Nagano, R. Baron, A. Sacco, A.M. Roccaro, O.C. Farokhzad, I.M. Ghobrial, Engineered nanomedicine for myeloma and bone microenvironment targeting, Proc Natl Acad Sci U S A. 111 (2014) 10287-10292. 27

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