(OA-72) Deciphering Myeloid Cell Dynamics within the Osteolytic Lesion Tumor Microenvironment to Overcome Immunosuppression in Multiple Myeloma

Understanding myeloid cells in the tumor microenvironment is crucial for identifying new targets against immunosuppressive barriers in multiple myeloma (MM). This study focused on neutrophils as potential targets for immune therapies by analyzing freshly isolated myeloid cells from focal lesions and bone marrow through single-cell RNA sequencing, imaging, and functional assays.

Methods: BM from healthy donors and BM and FL samples from 13 MM patients were freshly collected. CD11b+ myeloid cells were freshly isolated and selected cells were immediately used for scRNA-seq. In-vivo and in-vitro experiments conducted using MM models.

Results:

We assessed 105192 sorted CD11b+ cells from the MM TME in BM and FL sites, alongside 10086 cells from healthy BMs. Integration of these cells showed no new developmental clusters in MM-associated neutrophils, indicating a single developmental trajectory in both healthy and MM tissues. However, MM-associated neutrophils (TANs) exhibited distinct transcriptional profiles, forming three unique clusters enriched for CXCR2, differing from healthy donors. Inflammatory mediators were elevated in MM BM neutrophils, while immunosuppressive genes were higher in FL neutrophils.

We found that CXCR2+ neutrophils accumulated near malignant plasma cells in high tumor burden areas, particularly in FL using immunofluorescence. These findings align with scRNA sequencing data, showing more mature TANs with increased tumor burden. FL mature CXCR2+CD10+ neutrophils produced more chemokines involved in chemotaxis and migration and proinflammatory cytokines(CCL3 and CCL4) than those from MM BM and HD, suggesting a positive feedback loop enhancing CXCR2+ neutrophil recruitment in FL. FL mature TANs released more inflammatory and immunosuppressive cytokines, including IL-6,IL-1α compared to MM BM and HDM.Co-culture experiments revealed that CXCR2+CD10+ FL neutrophils suppressed T cell proliferation, indicating an immunosuppressive environment.

Using the VK*MYC murine model, we investigated the impact of CXCR2 inhibition on tumor growth. CXCR2 inhibitor reduced tumor burden and improved survival compared to controls, with synergistic effects when combined with BOR/DEX. In relapse, CXCR2 inhibition also decreased tumor burden and improved survival, showing synergistic benefits with triplets. Flow cytometry indicated increased anti-tumor cytokines, particularly with combination therapies.

Conclusions:

By analyzing BM and FL samples after sorting myeloid cells prior to sequencing allowed us to identify uncommon and/or labile subpopulations that are challenging to detect when sequencing entire CD45⁺ leukocyte populations. We discovered a maturation continuum from immature to a predominant population of mature TANs within FL. This suggests that neutrophils may mature and acquire an alternate development trajectory in FL. Targeting these neutrophils with CXCR2 inhibition reduces tumor burden and enhances survival, highlighting its potential as a therapeutic strategy in MM.