A Unique Crosstalk between Tumor Cells and Hematopoietic Stem Cells Reveals a Myeloid Differentiation Pattern Signature Contributing to Metastasis

Metastasis is the major cause of death in cancer patients. Recent studies have demonstrated that the crosstalk between different host and tumor cells in the tumor microenvironment regulates tumor progression and metastasis. Specifically, immune cell myeloid skewing is a prominent promoter of metastasis. While previous studies have demonstrated that the recruitment of myeloid cells to tumors is a critical step in dictating tumor fate, the reservoir of these cells in the bone marrow (BM) compartment and their differentiation pattern has not been explored.

Here we utilized a unique model system consisting of tumor cell clones with low and high metastatic potential (met-low and met-high, respectively) derived from melanoma and breast carcinoma cell lines. Hematopoietic stem cells (HSCs) and their early progenitor subset were defined as Lin-/Sca1+/CD117+, representing LSK cells. BM transplantation experiments using GFP-positive LSK cells derived from met-low and met-high tumor bearing mice were carried out to study lineage differentiation. The genetic signatures of LSK cells were analyzed by single cell RNA-sequencing (scRNA-seq). This analysis included unbiased automated annotation of individual cell types by correlating single-cell gene expression with reference transcriptomic data sets (SingleR algorithm) in order to evaluate the proportions of cell types in BM and reveal cell type-specific differentially expressed genes. Expression patterns of proteins originated from tumor cells were analyzed using a range of multi-omics techniques including nanostring, protein array, and mass spectrometry analysis. Tumor proteomic data was integrated with differential receptor expression patterns in BM cell types to reveal novel crosstalk between tumor cells and HSCs in the BM compartment.

Mice bearing met-high tumors exhibited a significant increase in the percentage of LSK cells in the BM in comparison to tumor-free mice or mice bearing met-low tumors. These results were confirmed by functional CFU assays of peripheral blood of met-high compared to met-low tumor bearing mice. In addition, mice that underwent BM transplantation with GFP-positive LSK cells obtained from met-high inoculated donors exhibited an increased percentage of circulating GFP-positive myeloid cells in comparison to counterpart mice transplanted with LSK cells from met-low inoculated donors. Moreover, scRNA-seq analysis of LSK cells obtained from the BM of met-low and met-high tumor bearing mice revealed that met-high tumors induce the enrichment of monocyte-dendritic progenitor population (MDP), confirmed also by flow cytometry. To uncover the possible factors involved in myeloid programming of LSK cells, we performed a proteomic screen of tumor conditioned medium and integrated the results with the scRNA-seq data analysis. This analysis revealed that the IL-6-IL-6R axis is highly active in LSK-derived MDP cells from mice bearing met-high tumors. An adoptive transfer experiment using MDP-GFP+ cells obtained from BM of met-high tumor bearing mice demonstrated that met-high tumors directly dictate HSC fate decision towards myeloid bias, resulting in increased metastasis. Evidently, blocking IL-6 in mice bearing met-high tumors reduced the number of MDP cells, and consequently decreased metastasis.

Our study reveals a unique crosstalk between tumor cells and HSCs. It provides new insight into the mechanism by which tumors contribute to the presence of supporting stroma. Specifically, tumors secreting IL-6 dictate a specific genetic signature in HSCs that programs them towards myeloid differentiation, thereby inducing a metastatic switch.