Targeting Myeloma-Associated Macrophage Inhibits Multiple Myeloma Progression By Enhancing Anti-Tumor Cytotoxic CD4⁺ T Cell Response

Background: Multiple myeloma (MM) is an incurable plasma cell cancer characterized by tumor cell accumulation and expansion in the bone marrow (BM). One of the major problems is that MM BM microenvironment is a tumor promoting and immune suppressive milieu. We previously discovered that macrophages (MФs), in particular myeloma-associated MФs (mMФs), heavily infiltrated into MM BM and mediated chemoresistance in MM. Colony-stimulating factor 1 receptor (CSF1R), also known as macrophage colony-stimulating factor receptor (M-CSFR), plays an important role as regulator of the development, morphology, survival, and functions of tissue macrophages as well as tumor-associated macrophages (TAMs). CSF1R blockade by inhibitors and antibodies has been shown promising to treat different tumors, such as glioma, pancreatic cancer, and diffuse-type giant cell tumor. We here assessed the impact of CSF1R blockade by CSF1R antibodies from Imclone (Lilly) on human and murine mMФs, MM growth in vivo, and anti-myeloma immune response in the BM microenvironment as well as chemotherapy in MM.

Methods: MФ infiltration was determined in the bone marrow (BM) patients with MGUS (n=6), MM (n=6), and compared to healthy donors (HD, n=6). CSF1R signaling blockade was assessed in the monocytes differentiation with M-CSF in the presence of CSF1R antibody or IgG control. CSF1R antibody impact on MФs growth/viability/proliferation was measured by trypan blue exclusion analysis, MTS, and Ki67 flow cytometry analysis. Tumor burden of 5TGM-1-bearing mice with CSF1R blockade treatment was determined by in vivo bioluminescent imaging, ELISA of IgG2b in mouse serum, and flow cytometry analysis of CD138⁺ myeloma cell infiltration in mouse BM. The effect of CSF1R antibody treatment on mMФ depletion and M1/M2 polarization was determined by flow cytometry and real-time PCR. Impact of CSF1R antibody treatment on cytotoxic CD8⁺ and CD4⁺ T cell immune response was measured by intracellular granzyme B flow cytometry and granzyme B ELISPOT. Effector cell-mediated MM cytotoxicity in the presence of mMФs with or without CSF1R treatment was measured by CD138/Annexin V flow cytometry. Survival rate of MM-bearing mice with CSF1R antibody and chemotherapy was evaluated using Kaplan-Meier estimates and log-rank tests using GraphPad Prism 5 software.

Results: MΦ accumulation in BM was associated with myeloma development. Blocking CSF1R by humanized and murine CSF1R monoclonal antibodies (CS4 and CS7) not only inhibited monocyte survival and differentiation but also suppressed human and mouse mMΦ survival and development in vitro. Further, Targeting of MФs by either CS7 antibody or DT-mediated MФ killing in Lysm^Cre X Csf1r^LsL-DTR C57BL/6 mouse had marked inhibitory effects on established myeloma progression. CSF1R blockade by CS7 treatment reprogrammed the tumor microenvironment toward to an anti-tumor phenotype. CSF1R antibody treatment reduced myeloma cell load in mouse BM, however the anti-MM activity by CSF1R antibody was abolished in immunodeficient Rag^-/- mice. Strikingly we found CSF1R blockade mediated anti-MM effect mainly based on CD4⁺ T cell response by CD8 and CD4 neutralizing antibody. Correspondingly cytotoxic anti-MM CD4⁺ T cell response enhanced by CSF1R antibody treatment was confirmed by ex vivo ELISPOT and in vitro cytotoxicity assay. Additionally, CSF1R antibody treatment promoted MM drug sensitivity.

Conclusion: Our data demonstrated thatMФs play an important role in MM growth and development. Targeting mMФs by CSF1R blockade achieves anti-MM activity by enhancing cytotoxic CD4⁺ T cell response and promotes chemotherapy on MM, therefore suggesting therapeutic strategies based on interfering with myeloma-macrophage interactions.