Associate professor ErasmusMC Cancer Institute, Netherlands
Introduction: The multiple myeloma (MM) bone marrow (BM) is characterized by IL-6 producing tumor-supportive inflammatory mesenchymal stromal cells (iMSC). iMSC are a source of neutrophil-modulation factors including IL6, C3 and chemokines for neutrophil-expressed CXCR1 and CXCR2. Since the BM permanently harbors large numbers of neutrophils, we hypothesized that stromal–neutrophil interactions might significantly impact the tumor-supportive BM environment in MM.
Methods: To investigate BM neutrophils in MM, we generated a single-cell transcriptomic overview of the entire neutrophilic lineage from fresh BM aspirates of 6 newly diagnosed MM (NDMM) patients, 6 MM patients that had completed intensive first-line treatment and four age-matched controls. In addition, single-cell RNA libraries were generated from BM MSC of 5 MM patients after intensive treatment. Functional interrogation of neutrophils and stromal cells included ex-vivo co-cultures, flow cytometric analyses, and RNA sequencing.
Results: Single-cell RNA-sequencing of the BM neutrophilic lineage in newly-diagnosed MM identified an increase in the percentage of mature neutrophils, and these cells were defined by inflammatory gene programs and genes associated with activation (DUSP1, GBP5), adhesion (ITGAX, ITGAL), and inflammation (OSM, CXCL8). Mature neutrophils in MM BM had an MM-supportive phenotype with increased transcription of the BCMA-ligand BAFF and the pro-inflammatory cytokine IL1B. In vitro co-cultures revealed that iMSC were sufficient to induce the MM neutrophil phenotype, including pro-inflammatory gene modules and production of BAFF and IL-1β. Neutrophil activation by iMSC was STAT3 dependent and could be abrogated by the STAT3-inhibitor Stattic. Reciprocally, iMSC-activated neutrophils gained the ability to induce MM-supportive iMSCs from non-activated stromal cells in an IL-1B-dependent manner. These data suggest that stromal cell-driven neutrophil activation could be involved in amplifying pro-tumor BM inflammation. To test whether this tumor-supportive inflammatory axis persists after first-line treatment, we performed single-cell RNA sequencing of neutrophils and MSC of patients that had completed induction- and consolidation therapy with triplet +/- anti-CD38 therapy, including high-dose melphalan and autologous stem cell transplant. iMSC were significantly reduced post-consolidation, but percentages remained elevated compared to controls. BM neutrophils, which were newly-formed post-transplant, had been re-activated and had re-acquired a pro-tumor phenotype. This was reflected in significantly elevated protein levels of BAFF and IL-1β in BM plasma of 52 patients after first-line treatment compared to non-cancer controls.
Conclusions: We identified an MM-supportive BM environment driven by MSC-neutrophil interactions that persist after treatment. Our data warrant investigation into novel strategies targeting both stromal and immune activation to disrupt the tumor-supportive BM environment in MM.
