Chemotherapy-induced reactive myelopoiesis leads to expansion of immunosuppressive neutrophil-like monocytes in mice and humans [scRNA-Seq]

Cytotoxic chemotherapy primarily targets rapidly proliferating cancer cells but also depletes normal myeloid cells. The resulting cell loss triggers reactive myelopoiesis, a compensatory process in which hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) regenerate myeloid lineages. We previously showed that the alkylating agent cyclophosphamide (CTX) induces myelopoiesis leading to the expansion of immunosuppressive monocytes in mice. However, the molecular features and clinical relevance of these cells remain poorly understood. Here, we report the emergence of immunosuppressive monocytes in the peripheral blood of lymphoma patients receiving CTX-containing chemotherapy. To gain mechanistic insight into CTX-induced myelopoiesis, we performed single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on BM monocytes from CTX-treated mice. These analyses revealed a heterogeneous monocyte population and demonstrated that CTX skews myelopoiesis toward the generation of neutrophil-like monocytes (NeuMo). Moreover, CTX-induced NeuMo cells, enriched within the CXCR4?CX3CR1? monocyte subset, exhibited potent T-cell suppressive activity. Using the NeuMo gene signature, reanalysis of public scRNA-seq datasets identified a transcriptionally similar monocyte subset in chemotherapy-treated cancer patients. Collectively, our findings suggest that the expansion of NeuMo-like cells following chemotherapy represents a conserved immunoregulatory feedback mechanism with potential impact on tumor response to chemoimmunotherapy. Overall design: For scRNA-seq of whole bone marrow cells, bone marrows were collected from cyclophosphamide-treated mice on day 2 (CTX_D2) and day 7 (CTX_D7). Bone marrows from untreated mice were used as the control (naïve). Live cells were enriched using the EasySep Dead Cell Removal kit (Stemcell Technologies). Equal numbers of cells from three individual mice under each condition were mixed and the pooled samples were used for downstream sequencing. For scRNA-seq of pre-enriched monocytes, bone marrow cells were stained with anti-CD11b and anti-Ly6C antibodies and sorted for CD11b+Ly6Chi monocytes by Bigfoot Spectral Cell Sorter. The purity of sorted monocytes was greater than 98%. Freshly isolated whole bone marrow cells or monocytes (CD11b+Ly6Chi) were processed for scRNA-seq libraries using the Chromium Controller (10X Genomics).