Mobilized peripheral blood: an updated perspective

Introduction

Discovered by pure chance in patients recovering from chemotherapy almost 45 years ago¹, the phenomenon of hematopoietic stem cell (HSC) mobilization has transformed the clinical practice of HSC transplantation². It has further extended to indications beyond HSC collection, including mobilization-based chemosensitization, conditioning, and gene therapeutic approaches, which are areas of intensive research. Better understanding of the pathways governing HSC trafficking can provide important insights into how stem cell localization within the bone marrow (BM) is regulated, which explains a continued need for basic research on mobilization to define the underlying molecular and cellular mechanisms.

In mammals, the first definitive HSCs arise in several intra and extraembryonic tissues from which they first migrate into the fetal liver^3,4. Following expansion in the fetal liver, HSCs continue their journey towards the BM, where the overwhelming majority of adult HSCs are subsequently found in their unique, specialized environments, the BM niches^5,6. Interestingly, despite the dramatically reduced migratory activity upon BM colonization, a small fraction of adult HSCs can be found in the peripheral circulation at any given time^7–9. Even though random leakiness of BM retention pathways cannot be excluded as a cause, the regularity of this physiological HSC egress^10–12 implies a biological function. The number of HSCs in the circulation at steady state can be substantially augmented by a wide variety of endogenous and exogenous stimuli such as growth factors^13–20, chemotherapy^1,21–23, chemokines^24–27, chemokine and integrin receptor agonists and antagonists^28–31, bioactive lipids^32,33, exercise^34,35, infection, and inflammation^36,37 (Figure 1). This enforced egress of HSCs into peripheral blood is called mobilization. While the function of homeostatically circulating HSCs remains enigmatic, pharmacologically induced HSC egress is increasingly used as the preferred strategy to generate grafts for HSC transplantation (HSCT), the only curative therapeutic option for many hematopoietic malignancies as well as non-malignant pathologies. HSCT requires the intravenous infusion of a minimum of 2×10⁶ CD34⁺ stem cells/kg recipient body weight; however, a dose of 5×10⁶ CD34⁺ cells/kg is considered preferable for early, consistent, and long-term multilineage engraftment^116–118. Each failure or delay to collect sufficient hematopoietic stem/progenitor cells (HSPCs) to proceed to transplantation extends the time of high-dose chemotherapy and increases the risk of disease progression in cancer patients.

Figure 1. Mobilization stimuli.

A wide variety of stimuli that lead to increased numbers of circulating hematopoietic stem cells (HSCs) have been identified, including but not limited to growth factors (cytokines¹⁷, granulocyte colony-stimulating factor [G-CSF]^19,38, granulocyte-macrophage colony-stimulating factor [GM-CSF]²⁰, stem cell factor [SCF]^18,39,40, FLT3 ligand [FLT3L]^40–42, thrombopoietin [TPO]^15,16,40, angiopoietin [Angpt]^43,44, vascular endothelial growth factor [VEGF]^43–45, and interleukins [ILs] -1⁴⁶, -3^47,48, -6⁴⁹, -7⁵⁰, -12⁵¹, -17⁵², and -33^53,54), chemotherapy-induced myeloid rebound^1,23,55, chemokines (CXCL12 and analogs^27,56, Gro-α⁵⁷, -β^26,58,57,59, and -γ⁵⁷, and IL-8^24,25,60), chemokine receptor antagonists (CXCR4 antagonists^61,62,63 and inhibitors of the intracellular mediator of CXCR4 signaling, the small Rho GTPase Rac1^64,65), bioactive lipids (sphingosine-1 phosphate [S1P]^66,67 and ceramide-1 phosphate [C1P]^66,67) and bacterial toxins^36,68,69 (lipopolysaccharide [LPS]^37,70 and pertussis toxin [PTX]^71–74), proteases (trypsin⁷⁵, matrix metalloprotease 9 [MMP9]^58,76, CD26^77,78, cathepsin G⁷⁹, and neutrophil elastase⁷⁹) and adenosine receptor agonists (defibrotide)^80,81, inhibitors of adhesive cell interactions⁸² (VLA4^28,29,83 and VLA4/VLA9⁸⁴ inhibitors, VCAM1⁸⁵, and CD44^86,87 blockers) and ephrin A3 receptor antagonists⁸⁸, polymeric sugar molecules (dextran⁸⁹, fucoidan^89,90, Betafectin PGG-Glucan⁹¹, and glycosaminoglycan [GAG] mimetics⁹²), and prostaglandin inhibitors⁹³. For the majority of the listed stimuli, a direct or indirect targeting of CXCR4 (green) or VLA4 (blue) signaling or both (red) has been documented. On the systemic level, the time of day^10,11,94, stress^95,96 and exercise^97–100, trauma and tissue damage^101–103, infection¹⁰⁴ and inflammation^105,106, coagulation^107–109 and complement^110–113 cascade along with cortisol^94,114 and the central¹¹⁴ as well as the sympathetic nervous system (SNS)¹¹⁵ have been shown to affect HSC egress out of the bone marrow (BM) into the peripheral blood. In sharp contrast to the diversity of mobilizing agents discovered and tested in preclinical models, only G-CSF alone (healthy donors) or in conjunction with chemotherapy and plerixafor (patients) is being used in the clinic. CNS, central nervous system.

The need to optimize mobilization regimens with regard to their stem cell yield, side effects and risk profile, cost-effectiveness, and availability for different groups of patients, as well as the need to better understand the communication between HSCs and their niche, continues to drive mobilization research. In this review, we discuss how deciphering the events induced by the most commonly used mobilizing agent, granulocyte colony-stimulating factor (G-CSF), led to the development of new mobilization strategies. We highlight the most recent findings and how we envision the newly discovered mobilization approaches will impact mobilization in the clinic. Alternative applications for mobilization are also reviewed. Lastly, we identify open questions and controversies, prospective directions, and how recent technical advances can be implemented within mobilization research.

Current mobilization regimens

G-CSF-mobilized blood is the preferred graft source for virtually all autologous and an increasing majority of allogeneic HSCTs owing to its generally higher stem cell content, reduced rates of graft failure, and better overall survival as compared to the BM^2,119,120. After 4–5 days of treatment with G-CSF, circulating HSPCs increase an average of 50–100-fold^121,122 as a result of HSPC pool expansion followed by mobilization. The latter is achieved through targeting the two major pathways involved in stem cell retention: chemokine receptor CXCR4- and integrin VLA4-mediated signaling^79,123,124. Attenuation of these pathways is achieved on the level of gene expression as well as through proteolytic cleavage^79,123–127. While the role of specific proteases involved in the latter, such as neutrophil elastase, cathepsin G, and MMP9, remains controversial^128,129, the cell surface protease dipeptidyl peptidase 4 (DPP-4, CD26), which cleaves and inactivates the CXCR4 ligand CXCL12, has indeed been shown to be essential for G-CSF-induced mobilization^77,78.

Shortcomings of G-CSF such as the slow mode of action^17,130, side effects, and contraindications^131,132 as well as significant heterogeneity in the mobilization response¹²¹ explain the quest for alternative mobilizing agents¹³³. Plerixafor (AMD3100), a small molecule bicyclam CXCR4 antagonist, is FDA approved for autologous stem cell mobilization in non-Hodgkin’s lymphoma and multiple myeloma (MM)^134,135. When combined with G-CSF, plerixafor increases CD34⁺ concentration 2–3-fold compared to G-CSF alone^134,135. However, a significant disadvantage of plerixafor is its cost, adding $25,567 per patient compared to G-CSF alone¹³⁶. Furthermore, up to 24% of patients undergoing autologous stem cell transplantation for lymphoma receiving plerixafor and G-CSF still fail to collect ≥2×10⁶ CD34⁺ cells/kg in 4 days of apheresis^134,135. A recent economic analysis determined that reducing apheresis by 1 day can potentially decrease medical costs by $6,600¹³⁷. Thus improved/alternative mobilizing agents and strategies are needed.

The long-standing view has been that both HSPC expansion and mobilization are necessary for clinically relevant mobilization. In line with this view, mobilization with CXCR4 or VLA4 antagonists alone fails to achieve numbers that would allow their use without G-CSF, despite promising potential in preclinical models^{29,61,83,138–140}. Very recent findings by our group and others challenge this notion and suggest that efficient recruitment of long-term, serially repopulating HSCs can be accomplished within minutes^57,58. Indeed, CXCR4 or VLA4 blockade, when combined with the stimulation of a different chemokine receptor, CXCR2, results in extremely rapid and potent HSC mobilization in mice with a repopulating capacity similar or even superior to G-CSF-recruited HSCs^57,58. These observations show that major changes in cellular composition or localization are not required for efficient mobilization. They further highlight the existence of different HSC species that, upon disruption of certain adhesive tethers, can egress from the BM very rapidly with kinetics that appear incompatible with a prior requirement for changes in gene expression.

The key role of the stromal compartment in G-CSF-induced mobilization has long been appreciated^124,129,141. Following activation of their G-CSF receptor, BM monocytes/macrophages, the most prominent hematopoietic component of the BM stroma^142–144, downregulate several retention molecules, including the major CXCR4 ligand, CXCL12, and several VLA4 ligands by non-hematopoietic stroma, resulting in HSPC egress^{143,145–147}. Absence of the G-CSF receptor on monocytes/macrophages abrogates the G-CSF-induced mobilization response¹⁴³, whereas the cytokine oncostatin M is thought to mediate communication between monocytes and non-hematopoietic stroma^145,148. Interestingly, rapid mobilizing agents in general, and chemokines and chemokine receptor antagonists in particular, are assumed to act on hematopoietic cells directly. Our recent findings challenge this view and suggest a critical contribution of stromal (endothelial) CXCR2 targeting upon rapid HSC mobilization by the combination of the CXCR2 ligand truncated Gro-beta (tGro-β) and a VLA4 antagonist⁵⁷. Since CXCR2 is absent from the HSPC surface^25,26, CXCR2-expressing neutrophils have long been assumed to be the responding cell. Upon stimulation with CXCR2 agonists, neutrophils release proteases that cut adhesive interactions between HSPCs and their niche^{60,76,149,150}. Yet CXCR2 within stroma was sufficient to induce mobilization with tGro-β and a VLA4 antagonist⁵⁷, pointing towards changes in endothelial layer permeability¹⁵¹ as well as crosstalk between endothelia and neutrophils as the “priming” events triggered by CXCR2 activation that boost VLA4 antagonist-induced HSPC egress (Figure 2).

Figure 2. Mobilization priming effects of CXCR2 stimulation.

Upon activation of the CXCR2 receptor on the surface of neutrophils (NE) and/or endothelial cells (EC), a reciprocal stimulation of the cells occurs that is critical for the subsequent boost of mobilization with a VLA4 or CXCR4 antagonist. Augmented mobilization appears to be a result of increased permeability of the endothelial layer together with other cell contact mediated or soluble factors derived from CXCR2-stimulated cells that reduce hematopoietic stem/progenitor cell (HSPC) retention. Additional inhibition of the VLA4 or CXCR4 receptor results in efficient targeting of the very primitive, serially repopulating HSPCs retained in the bone marrow (BM) primarily via VLA4 or CXCR4 signaling, respectively. The rapid kinetics of CXCR2 agonist + VLA4 or CXCR4 antagonist-induced mobilization preclude major molecular or cellular changes prior to the BM egress and rather suggest a close proximity of the primitive HSPC fraction to the BM sinusoids.

Development of alternative HSPC mobilization regimens and grafts

As we continue to learn about the events and mechanisms regulating HSPC egress, we approach the ultimate goal of developing an “optimal” mobilization strategy to collect sufficient numbers of primitive stem cells with superior properties within a day or two. In contrast to the days when clinical observations determined the applicability of a mobilization approach, educated and targeted designs are becoming the basis for the clinical development of mobilizing agents. In addition to the quantity and fitness of the HSPCs, as reflected in their engraftment capacity, the immunogenic properties of the graft (i.e. the graft-versus-host disease [GvHD] profile) are an important feature requiring optimization, potentially at the cost of stem cell numbers. For example, a substantially reduced incidence of GvHD is observed upon transplantation of CXCR4 antagonist-mobilized grafts, possibly due to co-mobilization of a specific population of dendritic cells (DCs) with immunomodulatory properties, plasmacytoid DCs^140,152,153. Along the same lines, grafts mobilized with pegylated G-CSF were superior to standard G-CSF in that they were associated with less GvHD^154,155 while graft-versus-leukemia (GvL) effects were improved through mobilization of invariant natural killer T (iNKT) cells¹⁵⁵. Susceptibility of the mobilized HSPCs to further molecular manipulation, e.g. using gene therapy, is another important criterion when defining the “optimal” mobilization strategy. Lastly, if proven to be suitable for mobilized HSPCs, recently described methods for ex vivo expansion of HSCs¹⁵⁶ are expected to shift the emphasis on HSPC quality over quantity even further.

Studies with CXCR4 and VLA4 antagonists, tested in VLA4 and CXCR4 knockout mice, respectively, implied an independence between the two axes^139,157,158. This suggests that subsets of HSPCs are being retained in the BM by either CXCR4 or VLA4. Combined with the knowledge of the complexity and multiplicity of events induced in the course of G-CSF mobilization^129,133, co-existence of these (and possibly other) functionally distinct HSPC populations suggests combinatorial mobilization approaches as the best alternatives to G-CSF. Thus, the small molecule Me6TREN reportedly inhibits CXCR4 and VLA4 signaling simultaneously, possibly through upregulation of the protease MMP9¹⁵⁹. However, given the controversy regarding the role of MMP9 for mobilization¹²⁸, other approaches should be explored. In addition to cell-intrinsic HSPC retention pathways, disruption of endothelial layer integrity, along with the endothelial cell activation and subsequent crosstalk between endothelial and mature hematopoietic cells, should be included in designing “optimal” mobilization. Recent data suggest that Viagra (sildenafil citrate), a phosphodiesterase type 5 (PDE5) inhibitor which blocks the degradation of cyclic GMP in the smooth muscle cells lining blood vessels, resulting in vasodilation, can synergize with plerixafor to rapidly mobilize stem cells in mice¹⁶⁰.

Various techniques for ex vivo graft manipulation (e.g. T cell depletion and CD34 enrichment^161–164) have been developed that entail extended periods during which the HSPCs stay outside of their natural environment and therefore, unsurprisingly, exhibit reduced stem cell capacity^165,166. From further in-depth analyses of differentially mobilized blood (see below), we expect to learn not only how to target specific HSPC populations but also how to mobilize HSPCs without a concurrent mobilization of mature cells, T-cells in particular. In general, a priori cell type-specific targeting remains challenging because of the high conservation of migratory and retention pathways between different hematopoietic cell types. Nevertheless, selective HSPC mobilization represents an intriguing goal that would help reduce additional graft manipulation.

Mobilization beyond stem cell collection

Open questions and future directions

Summary

Pharmacologically induced egress of HSPCs from the BM has become an indispensable tool in HSCT with all autologous and over 80% of allogeneic transplants performed with mobilized blood. Mechanistic insights gained from studying the complex chain of events induced during mobilization with G-CSF have paved the way for the rational design of alternative mobilization approaches without the inherent shortcomings of G-CSF such as slow mode of action and side effects. Similar to the recruitment of healthy HSPCs into the circulation, targeting of various BM retention pathways has been explored as a means to sensitize leukemic cells and thereby improve the efficacy of chemotherapy. Moreover, mobilization of HSPCs as a non-cytotoxic conditioning strategy as well as for gene therapy represents two other applications of mobilization beyond mere collection of a stem cell graft. Understanding the physiological function of homeostatic HSPC trafficking and identifying the genetic determinants of mobilization efficiency, along with characterizing the differentially mobilized HSPC populations on a single cell level, represent some of the directions of future mobilization research.