Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l -oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.

Collectively, these findings point to the importance of targeting SDF-1, and of neutralizing CXCR4 and CXCR7, both of which are expressed on tumor cells (). We reasoned that by neutralizing SDF-1, we can change the BM milieu, create a microenvironment that is less receptive for MM cells, diminish the homing of MM cells to the BM, and thereby inhibit MM disease progression. We report here on the discovery and characterization of olaptesed pegol (ola-PEG), a high-affinity-RNA Spiegelmer to SDF-1, and we demonstrate that ola-PEG-mediated neutralization of SDF-1 is a valid strategy for preventing MM progression in vivo.

The biological effects of SDF-1 are related its ability to trigger motility, chemotactic responses, adhesion, and the secretion of matrix metalloproteinases and angiopoietic factors. The importance of SDF-1 is also reflected by its ability to activate integrins such as LFA-1, VLA-4, and VLA-5 (expressed on HSCs), and to thus promote adhesion and migration (). Other factors such as fibroblast growth factors, transforming growth factor β, insulin-like growth factors I and II, platelet-derived growth factors, and bone morphogenetic proteins also contribute to the preferential homing of tumor cells to the BM. And additionally, it is likely that areas of high blood flow in red marrow facilitate tumor cell metastasis to these regions of BM (). Moreover, MM cells are characterized by the presence of CXCR4 (one of the SDF-1 receptors), and SDF-1-dependent signaling via CXCR4 plays a critical role in regulating fundamental biological functions of MM cells, such as migration and adhesion in vitro, as well as their homing to the bone marrow in vivo (). SDF-1 also activates the CXCR7 receptor (), which modulates trafficking and adhesion of human malignant hematopoietic cells.

Mechanisms that allow clonal multiple myeloma (MM) or solid tumor cells to home to and colonize bone marrow (BM) sites are not well defined. Seventy percent of patients with metastatic breast or prostate cancer present with involvement of the BM, and BM metastases also appear in about 15%–30% of patients with colon, lung, stomach, thyroid, prostate, and kidney cancer (). MM patients at diagnosis are characterized by the presence of multiple BM-lytic lesions, which indicate the perpetual dissemination of clonal plasma cells—originating in the birthplace of the MM clone—to multiple BM niches throughout the skeleton (); such lesions suggest that BM niches form an optimal environment for the lodgment and growth of MM cells. MM cells may also behave similarly to hematopoietic stem cells (HSCs), explaining why MM cells are selectively trafficked to BM sites (). In fact, it has been argued that clonal plasma cells—or other types of BM-metastasizing tumor cells—use the same signals and molecules that are critical for selection of the HSC niche: these include the chemoattractant stromal-cell-derived factor-1 (SDF-1/CXCL12), which is a major retention factor for HSCs and mature immune cells in BM tissue ().

The above findings were further corroborated at the protein level: primary MM BM-MSCs were pretreated with ola-PEG and subsequently cultured in the presence of MM cells; both MM and BM-MSCs cells were then exposed to ola-PEG or bortezomib, used alone or as a combinatory regimen. Ola-PEG inhibited the BM-MSC-dependent upregulation of p-ERK, p-Akt, and p-Src in MM cells, and also similarly affected Akt-downstream-targeted proteins ( Figure 6 D). Notably, ola-PEG treatment exerted a stronger effect when used in combination with bortezomib: in particular, ola-PEG was able to overcome the bortezomib-dependent activation of p-AKT, together with a more pronounced inhibition of Akt-downstream-targeted proteins (p-S6R; p-GSK3). In addition, proteins such as cofilin and paxilin, which are involved in the modulation of cell adhesion, were also modulated by the ola-PEG/bortezomib treatment ( Figure 6 D).

We next interrogated whether ola-PEG and bortezomib also act synergistically in modulating MM cell dissemination to distant BM niches in vivo, and, if so, whether this is due to the ability of ola-PEG to modulate cell adhesion. In vivo confocal microscopy revealed significant MM cell homing to the BM in control mice, whereas ola-PEG-treated mice presented with a lower number of MM cells within the whole skull BM area that was imaged ( Figure 6 B). These observations point to two possibilities: (1) that the BM of ola-PEG-treated mice is less receptive to MM cells, and (2) that the reduction in number of MM cells is due to an increase in mobilization of MM cells from the BM into the peripheral blood. Analysis of bortezomib-treated mice showed a significant reduction of MM cells, and this effect was even more significant with the combination of ola-PEG and bortezomib. These results were confirmed ex vivo, by quantifying the presence of MM cells in the femurs of treated mice ( Figures 6 C and S5 E) and were further supported by the demonstration of synergism between ola-PEG and bortezomib in inhibiting the adhesion of MM cells to primary MM BM-MSCs in vitro ( Figures S6 A–S6D).

When mice with established MM were treated with ola-PEG for 5 weeks, we observed reduced numbers of MM cells within the BM niches, along with increased numbers of MM cells in the circulation ( Figure S4 A). These results indicate that ola-PEG has an impact on the interaction of MM cells with the BM milieu in vivo, leading to mobilization of MM cells. We therefore evaluated the effect of ola-PEG as monotherapy, or as a combination therapy with bortezomib, on tumor progression and MM cell dissemination in vivo. Control mice, and mice treated with ola-PEG as a single agent, showed similar growth rates for MM.1S-GFP/Luc, whereas mice treated with a combination of ola-PEG and bortezomib presented with a significant reduction of tumor burden; this reduction was more pronounced when compared to mice treated with bortezomib as a single agent ( Figure 6 A). These data were supported by the finding that ola-PEG and bortezomib work synergistically in inhibiting MM cell proliferation in vitro, when tested on a panel of MM cell lines ( Figures S5 A–SD).

(D) Primary MM BM-MSCs were treated with ola-PEG for 10 hr and subsequently cultured in the presence of MM.1S cells for 8 hr. MM.1S cells were then harvested, and cell lysates were subjected to western blotting, with use of antibodies against p-ERK1/2, ERK1/2, p-cofilin, p-paxillin, p-Akt, Akt, p-Src, p-S6R, p-GSK3, and tubulin. MM.1S cells cultured in the absence of MM BM-MSCs were used as control.

(C) The presence of MM.1S-GFPcells ex vivo on femur tissues was detected by immunofluorescence. One representative image for one mouse from each group is shown (40×). For relative quantification, see Figure S5 E.

(B) Five weeks after MM cell inoculation, MM cell presence in BM structures of the skull was evaluated by intravital confocal microscopy (GFP + MM cells: green; Evans-Blue-positive blood vessels: red). Specific BM niches are highlighted, and relative magnification and 3D reconstruction are provided for each panel.

(A) SCID/Bg mice were injected (i.v.) with 5 × 10 6 MM.1S-GFP + /Luc + cells. Subsequently, mice were treated with vehicle (control), bortezomib (0.5 mg/kg, twice/week; i.p.), or ola-PEG (20 mg/kg, every other day; s.c.), alone or in combination (ola-PEG followed by bortezomib) (n = 5 per group). Tumor burden was detected by bioluminescence imaging, at different time points after MM cell injection (t0: second week; t1: fourth week; t2: fifth week). Error bars indicate SD. P indicates p value.

Because disruption of the SDF-1 gradient between BM and peripheral blood leads to the release of HSCs and lymphocytes into the circulation (), we argued that SDF-1 neutralization may lead to the release of MM cells from the BM into the peripheral circulation.

To understand the in vivo distribution and localization of ola-PEG in areas of MM cell growth, we injected (i.v.) mice that harbor MM.1S-GFPtumors with Alexa Fluor (AF) 647-conjugated ola-PEG and used live confocal imaging to determine the spatial localization of ola-PEG in BM niches that were colonized by MM cells; results documented that ola-PEG penetrated into BM areas that harbored MM cells ( Figure S3 C).

To better understand how BM niches are modulated by neutralizing SDF-1, we examined ola-PEG-mediated transcriptional changes in the BM microenvironment in vivo. SCID/Bg mice were treated with ola-PEG (s.c. injection, 20 mg/kg, given every other day) for 3 weeks. Untreated mice were used as controls. At the end of the third week, BM was harvested, and gene expression profiling was performed. Ola-PEG induced transcriptional downregulation of its target SDF-1, reduced expression of genes known to be regulators of angiogenesis, cell growth, cytokine production, vessel development and integrin-mediated cell adhesion, and downregulated protein kinase activity; in contrast, heat-shock proteins were upregulated ( Figure S3 B; p < 0.05). Genes were categorized using dChip software; see Supplemental Information for a detailed gene list.

To test the hypothesis that neutralization of SDF-1 in BM niches makes them less receptive to MM cell engraftment and colonization, we first examined whether ola-PEG-dependent SDF-1 inhibition prevents MM cell bone engraftment. MM cells were injected (i.v.) into control untreated mice, or into mice that were pretreated with ola-PEG (20 mg/kg; every other day) or with AMD3100 (5 mg/kg; daily; s.c.); treatment was continued after MM cell injection. Mice treated with ola-PEG showed significant reduction of MM cell tumor growth, relative to mice that were treated with AMD3100, or were untreated ( Figure 4 A), indicating that ola-PEG-dependent modulation of the BM milieu delays MM cell BM engraftment and tumor growth. Importantly, we detected differences in survival with ola-PEG-treated mice displaying significantly improved survival compared to those treated with AMD3100, or to untreated mice ( Figure 4 B). We next investigated whether ola-PEG treatment could also reduce MM cell dissemination from a primary BM site to distant bones. MM cell lines (murine or human) were loaded into bone chips and implanted subcutaneously (s.c.) into recipient mice that were pretreated with either vehicle control, or with ola-PEG. After the implantation, ola-PEG treatment was continued in the mice that were ola-PEG pretreated. Results showed that, compared to control mice, SDF-1 neutralization by ola-PEG significantly inhibited the colonization of distant BM sites by MM cells ( Figures 5 A–5D). Importantly, no ola-PEG-mediated cytotoxic effects were detected in MM cells in vitro ( Figure S3 A).

(A–D) Ola-PEG-dependent inhibition of SDF-1 inhibited in vivo colonization of MM cells (GFP + 5TGM1; GFP + MM.1S), originating at a primary bone marrow site, to distant bone niches, as demonstrated by flow cytometry (GFP + ) and immunostaining for murine (m) and human (h)-CD138. H.E. indicates hematoxylin-eosin staining (20×; 40×). Mice were euthanized when signs of paralysis were detected. Error bars indicate SD in (B) and (D). P indicates p values.

(A) Three weeks of pretreatment with ola-PEG (20 mg/kg injected every other day; s.c.) led to inhibition of MM tumor progression, shown with use of bioluminescence imaging. Ola-PEG-treated mice are compared to mice that were pretreated with AMD3100 (5 mg/kg, daily; s.c.), or to untreated mice (n = 8/group). Error bars indicate SD. P indicates p value.

Following a single subcutaneous (s.c.) administration of 10 mg/kg ola-PEG in mice, maximum plasma concentrations (C) of ola-PEG were observed at 12 and 6 hr in male and female mice, respectively (t) ( Table S2 Figure S2 D). Thereafter, the plasma concentrations of ola-PEG declined, with a mean apparent half-life (t) of 13 hr, which is comparable to the value obtained after intravenous (i.v.) administration. The bioavailability (F) of ola-PEG in male and female mice was estimated to be approximately 29.8% and 39.8%, respectively, with no appreciable or consistent gender-related difference in systemic exposure (AUC and C) to ola-PEG. Human pharmacokinetics and pharmacodynamics, along with safety evaluations, were performed in healthy volunteers in a clinical phase I study (): ola-PEG had a mean plasma half-life of 33–40 hr, and was safe and well tolerated up to the highest dose of 10.8 mg/kg, when administered as a 15 min i.v. infusion. ola-PEG mobilized white blood cells and hematopoietic stem and progenitor cells, as shown by CD34cell counts and colony formation assays.

We evaluated the influence of SDF-1 on the expression of CXCR4 and assessed whether SDF-1-induced activity could be blocked by ola-PEG. Stimulation of Jurkat cells with human SDF-1 resulted in a dose-dependent internalization of CXCR4. The maximal effect was reached at around 3 nM, half-maximal internalization was achieved at around 0.3 nM. Increasing concentrations of ola-PEG inhibited SDF-1-mediated CXCR4 receptor internalization, with an ICof 200 pM ( Figure 3 B). Also, ola-PEG inhibited SDF-1-mediated chemotaxis of CXCR4-expressing Jurkat cells in a dose-dependent manner, displaying an ICof 200 pM ( Figure 3 C). Additionally, using a β-arrestin complementation assay with a CXCR7 reporter cell line, we confirmed that ola-PEG also blocks SDF-1-dependent activation of the second SDF-1 receptor CXCR7. At a fixed concentration of 10 nM SDF-1 (EC), the half-maximal inhibition (IC) was at 5.1 nM ola-PEG ( Figure 3 D). (Note that for stoichiometric reasons, the assay does not allow measurement of ICvalues lower than 5 nM.) A nonfunctional Spiegelmer with the reverse sequence showed no effects (data not shown).

The RNA aptamer 193-G2-001 ola-PEG was converted into its corresponding Spiegelmer. A secondary structure prediction (mfold) is depicted in Figure 2 B (). To slow down renal clearance of the Spiegelmer, we increased its molecular weight by conjugating a 40 kDa polyethylene glycol (PEG) to its 5′ end. This PEGylated-RNA oligonucleotide was named 0p; all subsequent in vitro and in vivo data were generated with ola-PEG. Kof approximately 200 pM to human and mouse SDF-1α/β/γ were determined with use of surface plasmon resonance (Biacore) under physiological conditions ( Figure 2 C; Table S1 ). Because the 3D structures of chemokines are conserved (), we used a competitive assay format to test ola-PEG binding against a panel of 23 different chemokines. Other than soluble SDF-1, none of the other chemokines could compete with ola-PEG, demonstrating a high selectivity of ola-PEG to SDF-1 ( Figure 3 A).

(C) Inhibition of SDF-1-induced chemotaxis. Ola-PEG inhibits chemotaxis of Jurkat cells, with an IC 50 of approximately 200 pM. Baseline level (fluorescence measured without SDF-1) is reached at approximately 800 pM ola-PEG. Means ± SD for triplicate measurements are shown.

(B) Inhibition of SDF-1α-induced CXCR4 receptor internalization by ola-PEG. Jurkat cells were incubated with 0.3 nM SDF-1, plus various concentrations of ola-PEG. CXCR4 surface expression was quantified by flow cytometry, using a CXCR4 specific PE-labeled antibody. Data points are means ± SD for triplicate measurements. ola-PEG inhibits CXCR4 internalization with an IC 50 of approximately 200 pM.

(A) CC, CXC, CX3C, and XC chemokines (2 μM) were checked for competing with the binding of ola-PEG (12.5 nM) to immobilized human SDF-1α. As expected, mixtures of ola-PEG with SDF-1α or SDF-1β competed fully, but none of the other chemokines did so. Data are double referenced and are plotted as mean response units ±SD of n = 2 injections.

For in vitro selection against-SDF-1, we used reiterative rounds with increasing stringency and amplification steps that included mutagenic PCR (in rounds 15 and 17; Figure S2 A). The enriched library from round 18 was cloned and sequenced. An alignment revealed sequences that differed by point mutations and by a nonrelated orphan sequence ( Figure S2 B). After truncating the primer binding sites, the best aptamer (193-G2) was trimmed to a 45-mer (193-G2-001) without loss of affinity ( Figure S2 C). A pull-down assay showed that the binding of 193-G2-001 to-SDF-1 resulted in a dissociation constant (K) of 268 pM at 37°C ( Figure 2 A).

(B) Secondary structure prediction for olaptesed, with potential base pairs forming three hydrogen bonds depicted in red, and those forming two hydrogen bonds with each other shown in blue.

We identified a high-affinity, structured-RNA oligonucleotide (Spiegelmer) () that binds and inhibits SDF-1. Initially, we applied an in vitro evolutionary screening technology based on the SELEX process () to select aptamers from a pool of randomized RNA oligonucleotides against-SDF-1 (the enantiomer of natural-SDF-1). This process led to the identification of aptamer sequences, which were synthesized as Spiegelmers by using nonnatural-ribonucleotides. The Spiegelmers were then able to bind to natural-SDF-1.

To better define the functional role of MM BM-MSC-secreted SDF-1 on MM cells, we used primary MM BM-MSCs to undertake SDF-1 loss-of-function studies. Transduction and knockdown efficiency of SDF-1 were evaluated with use of immunofluorescence and ELISA, respectively ( Figures S1 A and S1B). Results showed that knocking down the secretion of SDF-1 from MM BM-MSCs inhibits the adhesion and also the migration of MM cells toward primary MM BM-MSCs ( Figures S1 C and S1D). Moreover, MM cells cultured in the presence of BM-MSC cells in which SDF-1 was knocked down presented with reduced activation of prosurvival and migration-related pathways ( Figure S1 E).

We next focused on corroborating the role of SDF-1 in facilitating the homing, lodgment, and growth of MM cells in vivo. We used in vivo live confocal imaging to determine the localization and spatial distribution of SDF-1 in BM niches that are occupied by MM cells. By injecting a fluorescently labeled anti-SDF-1 in mice that harbor MM GFPtumors, we found that SDF-1 was enriched in BM areas that were colonized by MM cells, whereas SDF-1 was only observed within blood vessels in BM niches that were free of MM cells ( Figure 1 B). This observation is consistent with prior reports that transplanted hematopoietic stem and progenitor cell populations localize in subdomains of BM microvessels, where the SDF-1 chemokine is particularly abundant (). The finding was also supported by the significantly higher level of SDF-1 secreted from primary BM-mesenchymal stromal cells (MSCs) that were obtained from MM patients, relative to those that were harvested from healthy subjects ( Figure 1 C). Taken together, these results demonstrate that MM cells preferentially localized in SDF-1-enriched BM niche.

Given the importance of SDF-1 in facilitating bone metastasis in a variety of clonal tumor cells, we compared the expression of this chemokine in the BM of patients with bone metastases of solid tumors or of multiple myeloma (MM), to that in the BM of healthy individuals or of patients with monoclonal gammopathy of undetermined significance (MGUS). Our findings indicate that BM that is affected by colonization of solid tumor cells or MM cells presents with higher expression of SDF-1, compared to samples from healthy controls or from MGUS patients, which showed scattered, minimal SDF-1 expression ( Figure 1 A). These findings indicate the significance of SDF-1 in the BM niche of metastatic tumors, including MM as a model of BM metastasis.

(B) SCID/Bg mice were injected (i.v.) with 5 × 10 6 MM.1S-GFP + cells; after 3 weeks, Alexa Fluor (AF) 633-conjugated anti-SDF-1α (Ab) was administered i.v., and mouse skull BM niches were imaged after 4 hr, using in vivo confocal microscopy. Evans Blue was used to visualize blood vessels (MM.1S-GFP + /Luc + cells: green; vessels: red; AF633-anti-SDF-1α Ab: blue).

(A) Bone marrow (BM) specimens were obtained from patients with multiple myeloma, MGUS, or solid tumors and stained with anti-SDF-1; MGUS samples were double-stained for SDF-1 (brown) and CD138 (blue). Normal BM was used as control. P indicates p value.

Discussion

Ebos et al., 2009 Ebos J.M.

Lee C.R.

Cruz-Munoz W.

Bjarnason G.A.

Christensen J.G.

Kerbel R.S. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. The development of therapeutic agents that prevent or delay metastasis has been hampered by many limitations and failures; indeed, a number of antineoplastic agents that are effective on primary tumor sites do not display the same ability to inhibit the proliferation of disseminated cancer cells, and there is a pressing need to develop novel therapeutic strategies that disrupt the permissive metastatic niche and thus reduce the capacity of cancer cells to home to and engraft in these areas. Agents such as bisphosphonates, anti-RANK, and TGF-β inhibitors such as LY2157299 show promising activity, but none demonstrates significant clinical activity in preventing or delaying metastases. Antiangiogenic molecules are implicated in inhibiting the process of metastasis; however, recent evidence shows that such drugs may in fact paradoxically increase metastatic potential, due to the induction of hypoxia within the tumor mass ().

Here, we use MM as a model of bone-to-bone cell dissemination. We first demonstrate that SDF-1 colocalizes in areas of the BM where metastasis occurs, not only in multiple epithelial tumors, but also in advanced stages of MM. Indeed, by comparing early precursor stages (such as MGUS) to late, disseminated stages of advanced MM, we were able to document a significant increase in SDF-1 levels in the BM with advanced disease. We also report on the identification and characterization of ola-PEG, a PEGylated l -RNA Spiegelmer that binds the chemokine SDF-1 with high affinity and selectivity. Our cell-based studies show that ola-PEG potently inhibits SDF-1 action, and thereby affects downstream signaling in tumor cells. Most interestingly, our data show that ola-PEG-dependent neutralization of SDF-1 modulates the premetastatic BM niche in vivo and thus inhibits MM tumor progression and prolonged survival, compared to AMD3100-treated mice. SDF-1 inhibition also affects the BM gene expression signature by altering regulators of angiogenesis, adhesion, and proliferation that collectively modify the BM niche; significantly reduces the homing of injected MM cells to the BM of SCID/Bg mice; and decreases MM cell bone metastases in vivo. Whether these effects are due to inhibition of SDF-1 signaling in the tumor cell itself, or due to changes in the stroma and its associated gene expression, has yet to be determined. Nevertheless, our findings clearly document a therapeutic effect of ola-PEG in a MM xenograft model of established disease. Ola-PEG colocalizes in areas of high tumor burden within the BM and disrupts the interaction of MM cells with the BM niches, as evidenced by its influence on diminishing tumor mobilization, homing, and growth within the BM niches. In addition, ola-PEG also chemosensitizes MM cells to bortezomib, despite the fact that it (ola-PEG) has no single-agent activity on the tumor cells, indicating that the activity of ola-PEG is specific to the niche and its interaction with SDF-1. Thus, combinations of ola-PEG with other chemotherapeutic agents are also likely to lead to synergistic effects.

In conclusion, our data suggest that the anti-SDF-1 Spiegelmer ola-PEG represents an agent that successfully targets the interaction between BM niches and tumor cells, thereby preventing or disrupting BM colonization by MM cells and potentially also by other bone-metastasizing tumor cells.