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M.N. Zeissig et al.
CXCL12, produces abundant CCL3, suggesting that endogenous CCL3 expression can suppress response to CXCL12. Notably, migration of U266 cells towards CXCL12 could be restored by either CCR1 KO or treatment with a CCR1 inhibitor.17 Here, we observed that treatment of OPM2-EV-1 control cells with CCL3 prevented migra- tion towards CXCL12, in accordance with our previous study,17 whereas, the chemotactic response of OPM2- CCR1-KO-1 cells to exogenous CXCL12 was maintained following CCL3 treatment. These data strongly suggest
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that CCL3/CCR1 signaling is responsible for blocking migration towards CXCL12 in these cell lines. Notably, CCL3/CCR1 signaling has been shown to drive mobiliza- tion of hematopoietic progenitors and natural killer cells from the BM in part through inactivation of CXCR4.25,26,37 Based on these studies, we postulated that CCL3/CCR1 signaling may inhibit the CXCL12 mediated retention of MM PC in the BM, enabling the egress of MM PC into the circulation and subsequent dissemination. Indeed, we demonstrate here that CCR1 expression increases the
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Figure 7. CCR1 inhibition reduces circulating multiple myeloma plasma cell numbers and tumor dissemination in NSG mice bearing OPM2 or RPMI-8226 cells. (A) Primary tumor burden in injected tibiae after 4 weeks in NSG mice injected with OPM2-EV-1 cells and treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12- hour intervals. (B) Primary tumor burden in injected tibiae after 4 weeks in NSG mice injected with RPMI-8226-luc cells and treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12-hour intervals. (C) Number of circulat- ing OPM2-EV-1 cells in peripheral blood of mice treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12-hour intervals. (D) Number of circulating RPMI-8226-luc cells in peripheral blood of mice treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12-hour inter- vals. (E). Tumor burden disseminated to the non-injected contralateral leg in mice injected with OPM2-EV-1 cells treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12-hour intervals. (F) Tumor burden disseminat- ed to the non-injected contralateral leg in mice injected with RPMI-8226-luc cells treated days 3-28 with CCX9588 (15 mg/kg) or vehicle control at 12-hour intervals. (G) Spleens collected from naïve NSG mice or vehicle- or CCX9588-treated mice bearing OPM2-EV-1 or RPMI-8226- luc cells were measured to assess the degree of splenomegaly. Naïve mice splenic sizes are duplicated from Figure 5D for comparison. Box and whisker plots depict median and interquartile range, n=10-12 mice/group (A, C and E), n=17 mice/group (B, D and F), n=7-17 mice/group (G). **P<0.01, ***P<0.001, ****P<0.0001, Mann-Whitney test (C, E and F), ordinary one-way ANOVA with Tukey’s multiple
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haematologica | 2021; 106(12)