High-Resolution Single-Cell RNA Sequencing Uncovers Disparate Neutrophil Populations within Metastatic Cancer Lesions Linked to Progression Free Survival in Multiple Myeloma Patients

Understanding the myeloid cell biology of the tumor microenvironment (TME) will allow discovery of novel strategies to disrupt the local immunosuppressive milieu and to develop effective immune based therapies. Neutrophils are a major, understudied myeloid cell population in metastatic malignant tumors. Here, we studied myeloid cells freshly sorted from paired focal lesions (FL) (osteolytic lesions and plasmacytomas) and pelvic bone marrow (BM) from multiple myeloma (MM) patients. Using single cell RNA sequencing and functional assays, we discovered the emergence and expansion of distinct, late-stage neutrophil subsets with potent immunosuppressive signatures predominantly in FL. The most abundant late-stage neutrophil subset, MM_N_FL1, activates cytokine responses, the IL-8 CXCR2 pathway, and nitrogen compound metabolism while downregulating primary, secondary, and tertiary granule expression. FL neutrophils exhibit transcriptional and functional differences compared to their paired BM counterparts, displaying notably elevated immunosuppressive capacities independent of tumor cell percentage in FL and BM. Furthermore, the gene signature of the FL neutrophils was associated with significantly inferior progression-free survival (PFS) in a defined population of MM patients. The study provides an in-depth analysis of neutrophil dynamics, function, and imbalance in MM patient FL, suggesting promising targets for novel clinical immunomodulatory therapies. Overall design: A total of 13 patients underwent biopsy: 10 had osteolytic lesions (OL) and 2 had extramedullary disease. These are grouped together as FL because the genetic analysis was the same for both populations. One OL had no cells recovered. We also included three healthy donors as basline control for patients. BM from healthy donors and BM or FL samples from MM patients were freshly collected using either standard blind aspiration (from healthy volunteers) or CT guided biopsy and aspiration (from patients) at the Roswell Park Comprehensive Cancer Center. The study was approved by the Roswell Park Institutional Review Board (protocol # I 66418). Informed consent was obtained from all patients and healthy volunteers before sampling. Ammonium–chloride–potassium (ACK) lysing buffer was used to exclude red blood cells (RBCs). CD11b+CD3-CD56-CD138- myeloid cells were freshly isolated using a SONY flow sorter. Selected cells were immediately used for scRNA-seq.