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CCR1 drives dissemination of multiple myeloma plasma cells
extravasation and homing to another BM site. Integrin mediated adhesion of MM PC to BM stromal cells (BMSC), and extracellular matrix components synthesized by BMSC, is well-established to mediate retention of MM PC within the niche.13 For example, MM PC express the inte- grin α4b1 (also known as very late antigen 4, VLA-4) that mediates adhesion to vascular cell–adhesion molecule 1 (VCAM-1) on BMSC and to the extracellular matrix com- ponent fibronectin.13 Importantly, the C-X-C chemokine ligand CXCL12 (also known as stromal cell-derived factor- 1; SDF-1), abundantly produced by BMSC,14 enhances adhesion to fibronectin and VCAM-1 through binding to its receptor CXCR4 on the surface of MM PC and inducing rapid conformational changes of the integrin α4b1 com- plex on MM PC.15 Notably, plerixafor-mediated inhibition of the CXCL12 receptor CXCR4 on MM PC results in mobilization of MM cells to the peripheral blood (PB) in a preclinical model of MM.15 These data suggest that CXCL12 is a critical BM retention signal for MM PC and that overcoming the CXCL12/CXCR4 signal may be required for release from the niche during dissemination.
In a previous study by Azab and colleagues, increased hypoxia in the BM was shown to be associated with an increase in circulating MM PC in a preclinical model.16 Additionally, we have previously identified that overex- pression of the hypoxia-inducible factor 2α (HIF-2α) in MM cell lines reduces their response to exogenous CXCL12 in vitro, suggesting that hypoxia may overcome CXCL12-mediated retention. Furthermore, we identified that hypoxia and HIF-2α increased expression of the C-C chemokine receptor CCR1 in human MM cell lines.17 CCR1 is a seven-transmembrane G-protein coupled recep- tor and its most potent activator is CCL3 (also known as macrophage inflammatory protein 1α; MIP-1α). Previous literature suggests that MM PC abundantly produce CCL318-21 which activates CCR1 expressed on osteoclasts leading to increased osteolysis,19 with CCR1 antagonists reducing osteolysis in a murine model of MM.22,23 In addi- tion, CCL3 is a potent inducer of migration of patient- derived MM PC and MM cell lines in vitro.17,19,20,24 In hematopoietic progenitors and natural killer cells, CCL3/CCR1 signaling drives mobilization from the BM, in part by inactivation of CXCL12/CXCR4.25,26 Similarly, our previous studies showed that either pre-treatment of MM cell lines with CCL3 or elevated CCR1 expression decreased tumor cell migration towards CXCL12 in vitro.17 Taken together, these data suggest that hypoxia-mediated increases in CCR1 expression may desensitize cells to CXCL12-mediated BM retention and thereby facilitate dis- semination. In support of this, we have previously shown that expression of CCR1 in MM PC is associated with poorer prognosis and an increase in the number of circulat- ing MM PC in newly diagnosed MM patients.17 Here, we further investigated the association between CCR1 expres- sion and poor overall survival rates in MM patients. Furthermore, we investigated the role for CCR1 in the dis- semination of MM PC in vivo. Initially, we determined whether CCR1 overexpression can promote tumor dis- semination in the syngeneic 5TGM1/KaLwRij murine model of MM. Furthermore, using xenograft models of MM, we assessed whether CCR1 knockout limits the dis- semination of MM PC in vivo. Lastly, we investigated whether pharmacological inhibition of CCR1 can be used as a viable therapeutic strategy to limit MM PC dissemina- tion.
Methods
Flow cytometry on patient samples
Ethical approval for this study was obtained from the University of Freiburg Medical Center Ethics Review Committee and all patients provided written, informed consent, in accordance with the Declaration of Helsinki. CCR1 analysis was conducted on BM mononuclear cells from BM aspirates from 28 newly diag- nosed MM (median age: 68 years [range, 49–84]; male:female ratio 1.15:1) and seven monoclonal gammopathy of undetermined sig- nificance (MGUS)1 (median age: 74 years [range, 53-88]; male:female ratio 1.7:1) patients. Cell surface CCR1 expression was assessed on viable CD38++/CD138+/CD45lo/CD19- malignant PC by multicolor flow cytometry (FACSARIA III; BD Biosciences, San Jose, CA) as previously described.17
Murine multiple myeloma models models
C57BL.KaLwRijHsd (KaLwRij) mice were inoculated into the left tibia with 1x105 5TGM1-CCR1 or 5TGM1-EV cells. After 25 days, splenic tumor burden was assessed by bioluminescent imag- ing (Xenogen IVIS 100; Perkin Elmer), and tumor burden in the PB, injected tibiae, and pooled tibiae and femora from the contralater- al leg was assessed by flow cytometry (LSRFortessa flow cytome- ter).
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were inoculated intratibially with 5x105 OPM2-CCR1-KO-1 or OPM2-EV-1 cells. For CCX9588 studies, mice were treated at 12-hour intervals via oral gavage with either the CCR1 antagonist CCX9588 (15 mg/kg; ChemoCentryx, CA) or polyethylene glycol (PEG) vehicle alone, commencing day 3 or day 14 following tumor cell injection. Primary and secondary BM and splenic tumor burden and PB tumour cells were assessed 28 days after tumor cell injection.
Detailed methods can be found in the Online Supplementary Methods.
Results
High CCR1 expression is associated with poorer prognosis in multiple myeloma patients
We used flow cytometry to examine the expression of CCR1 on CD38++/CD138+/CD45lo/CD19- BM PC in a cohort of MM and MGUS patients who had not received previous treatment. BM PC expression of CCR1 was detectable by flow cytometry in 14.3% (one of seven) of MGUS patients and in 53.6% (15 of 28) of MM patients (Figure 1A). Furthermore, BM PC expression of CCR1 was significantly higher in MM patients than in MGUS patients (P<0.05, Figure 1A), consistent with our previous microar- ray analysis.17
Our previous analysis suggested that high levels of CCR1 expression in BM PC from newly diagnosed MM patients was associated with poorer overall survival in patients enrolled in the total therapy 3 trial.17 Here, we performed Cox regression analysis to determine if elevated (above median) CCR1 expression was an independent predictor of poor prognosis. In univariate analyses, elevated CCR1 expression, high-risk gene expression signature, elevated serum b2 microglobulin (≥5.5 mg/L), anemia (hemoglobin < 100 mg/L) and high plasma cell proliferative index (PI ≥10%) were associated with inferior overall survival (P<0.05, Table 1). Notably, multivariable analysis demon- strated that elevated CCR1 retained its association with poor prognosis (P<0.05, hazard ratio [HR]=2.5, 95% confi- dence interval [CI]: 1.0-5.9), when these other prognostic
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