Chemokines CCL14 and CCL3 Facilitate Monocytes/Macrophage Infiltration in Multiple Myeloma Bone Marrow
Authors: Li Yi , Zheng Yuhuan , Yang Li , Wang Qiang , Bi Enguang , Li Tianshu , Lu Yong , Qian Jianfei , Zhang Mingjun , Yang Maojie , Yi Qing , Cai Zhen 
Published: November 2019

Abstract
Drug resistance is the most common reason for treatment failure in multiple myeloma (MM), a plasma cell cancer of the bone marrow (BM). Our previous studies have shown that macrophage (MΦ) infiltration is increased in MM BM, and these MM-associated MΦs induce MM drug resistance by protecting MM cells from chemotherapeutic-induced cell death. A recently published clinical study also suggests that the number of BM MΦs negatively correlates with MM drug response and patient survival. Why MM BM harbors more MΦ is not well understood. In general, MΦs are derived from circulating monocytes, and monocytes are recruited to tissues by chemokines and differentiate into MΦs. In this study, we examined monocyte/MΦ chemokine expression in MM BM and determined that CCL14 and CCL3 were functional chemokines that regulated monocyte/MΦ infiltration in MM BM.
To identify chemokines that regulate monocyte/MΦ infiltration in the MM tumor bed, we examined a panel of chemokines expressed in MM patient BM cells. Specifically, total BM cells (both CD138+ and CD138- cells) from MM patients were assayed by qPCR for target gene expression analysis. MM BM cells highly expressed CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CCL14 (HCC-1), but not CCL8 (MCP-2), CCL7 (MCP-3) or CCL13 (MCP-4). Next, we compared expression of those chemokines in MM BM vs. healthy BM aspirates by ELISA. MIP-1 α and HCC-1 were highly expressed in MM BM aspirates (n=11), but not in BM from healthy donors (n=7; P<0.05). Immunohistochemistry staining also confirmed that MM BM (n=5 patients) highly expressed MIP-1 α and HCC-1. Based on our findings, we hypothesized that elevated MΦ infiltration in MM BM might be due to MM BM overexpression of MIP-1α and HCC-1. To test this hypothesis, we first examined MIP-1α and HCC-1 function in monocyte migration. In vitro chemotactic assay showed that adding MIP-1α or HCC-1 neutralizing antibody inhibited monocyte migration to cocultured BM stromal cells (BMSCs) and MM cells (P<0.05). The antibodies also inhibited monocyte migration to ex vivo cultured BM cells from MM patients (P<0.05). In a murine MM mouse model, C57BL/KawRij mice inoculated with 5TGM1, a murine MM cell line, developed tumors in hind leg bones. Both flow cytometry analysis and immunohistochemistry staining suggested that the tumor-bearing mice had an increased number of MΦs in the BM, compared with healthy mice (P<0.05). Because HCC-1 does not have murine homology but it has the highest amino acid sequence similarity to MIP-1α, we treated the mice with MIP-1α neutralization antibody. Intra-peritoneal injection of mouse MIP-1α antibody decreased the MΦ number in BM (P<0.05). Such in vivo findings strongly suggest that MIP-1α regulates MΦ infiltration in MM BM. We also examined the source cells that contributed to high MIP-1 α and HCC-1 expression in MM BM. Primary MM cells (CD138+) and non-malignant BM cells (CD138-) were isolated from patient (n=5) BM aspirates. qPCR analysis showed that both CD138+ and CD138- cells had high MIP-1α and HCC-1 expression. Further, BMSC/MM coculture stimulated MIP-1α overexpression in BMSCs. Finally, we examined the association between MIP-1α or HCC-1 expression and the number of BM MΦ in MM patients. The chemokine expression in BM aspirates was determined by ELISA, and the number of MΦ was measured by flow cytometry analysis for CD14+/CD68+ cells. We found that MIP-1α expression was positively associated with the number of BM MΦs (P<0.05).
To summarize, our findings suggest that CCL14 and CCL3 facilitate monocyte/MΦ infiltration in MM BM. Inhibiting these 2 chemokines may decrease the number of MM-associated MΦs, therefore increasing MM cell vulnerability to chemotherapy.

Disclosures
No relevant conflicts of interest to declare.