Haematopoiesis and tumour growth
During haematopoiesis, multilineage blood cells are generated successively from a series of lineage-restricted progenitor cells, in turn, derived from haematopoietic stem cells. Haematopoietic stem and progenitor cells (HSPCs) are usually localised in the bone marrow niche during the lifetime but extramedullary haematopoiesis can occur in organs like the spleen during pathophysiological conditions like cancer.
Immunosuppressive myeloid cells like myeloid-driven suppressor cells (MDSCs), tumour-associated macrophages (TAMs) and tumour-associated neutrophils (TANs) that are associated with tumour progression, enhance cancer cell stemness, invasion and immune escape. During expansion, tumours interfere with normal haematopoiesis in the bone marrow, promote splenic extramedullary hematopoiesis and induce the generation of myeloid cells with tumour-promoting functions. It is therefore very important to study the generation of HSPCs and understand their populations.
Studying HSPC generation
In this datasheet, the authors have used a melanoma mouse model to study tumour-driven perturbations during haematopoiesis. First, they harvested cells from the spleen of tumour-burdened mice and the bone marrow of tumour-burdened and healthy mice. From these cells, they performed enrichment of negative lineage (Lin-) cells by magnetic enrichment using the BD IMag™ Haematopoietic Progenitor Cell Enrichment Set, supplemented with biotinylated antibodies for CD2, CD5 and CD19. Enriched Lin- cells from tumour-burdened bone marrows and spleens, and healthy bone marrows were individually stained with unique DNA-barcoded antibodies from the BD Mouse Immune Single-Cell Multiplexing Kit and then stained with a 6-color antibody fluorochrome panel and a 36-plex BD® AbSeq Panel. Four different populations from each tissue were sorted simultaneously using the BD FACSMelody™ Cell Sorter with 4-way sorting capability (12 tubes total). Each one of the four populations from each tissue was pooled into a single tube (4 tubes total). Next, tubes were pooled and then loaded separately onto four cartridges using the BD Rhapsody™ Single-Cell Analysis System for single-cell capture. After sample retrieval, the BD® AbSeq, Sample Tag and mRNA (BD Rhapsody™ WTA) Libraries were prepared for sequencing. The sequencing results from ~31,000 cells were analyzed using SeqGeq™ v1.6 Software.
Results and conclusions
The 6-colour fluorochrome labelled antibody panel enabled the separation of HSPCs into Lin-Sca1+Ckit+ (LSK), common myeloid precursor (CMP), megakaryocyte erythroid progenitor (MEP) and granulocyte macrophage progenitor (GMP) cells. The 4-way sorting strategy of the BD FACSMelody™ enabled the purification of the fractions from the tumour-burdened (bone marrow and spleen) and healthy (bone marrow) samples. From the data, it could also be concluded that the spleens of the tumour-burdened mice had enrichment of HSPCs compared to those of healthy mice. Further, the data demonstrate the power of a comprehensive workflow integrating the BD FACSMelody™ Cell Sorter and BD Rhapsody™ Single-Cell Analysis System for deep characterization of rare cell types and the potential discovery of signatures associated with altered cellular processes.
The study, data and analysis can be found in this downloadable datasheet.
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