Neutrophils are the most abundant type of circulating immune system cell, and they act as the body’s earliest form of defense against infection. We know that neurophils originate in the bone marrow (BM), but scientists haven’t, to date, been able to find a progenitor that only gives rise to this type of white blood cell. Researchers at La Jolla Institute for Allergy and Immunology have now used advanced biomarker analysis and RNA sequencing techniques to identify neutrophil-specific progenitors in the bone marrow of mice and humans, which appear to directly promote tumor growth in mice, and which are found in high numbers in the blood of human cancer patients. 

The team suggests that the findings offer new insights into the biology of neutrophil progenitors, which should help to develop new targets for neutrophil-related disorders, including cancer, and potentially lead to simple diagnostic tests. “We found that this particular subset of progenitor cells promotes tumor growth and shows up in high numbers in melanoma patients,” comments research lead Catherine Hedrick, Ph.D., a professor in the division of inflammation biology. “If our initial findings hold up across the board, a simple blood test could indicate if you have cancer or not.”

The studies are described in Cell Reports, in a paper titled, “Identification of an Early Unipotent  Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow.”

Neutrophils are the most abundant type of circulating immune system cell, and are quickly mobilized to arrive at the site of new infection. Studies suggest that these short-lived cells play critical roles in chronic inflammatory diseases including cancer, the authors write. Like other blood and immune system cells, neutrophils are produced in the bone marrow, from stem and progenitor cells. Multipotent hematopoietic stem cells in the bone marrow give rise to increasingly specialized sets of progenitor cells that finally differentiate into the different blood cell types, including red blood cells, lymphocytes, and neutrophils. It’s already known that neutrophils and monocytes both originate from granulocyte monocyte progenitor (GMP) cells but further stages in the line of differentiation is less clear, and a dedicated neutrophil-specific progenitor hasn’t been identified “unipotent neutrophil progenitors are not well defined,” the authors write.

Knowledge so far indicates that the bone marrow comprises populations of common myeloid progenitors (CMP), megakaryocyte erythroid progenitors (MEP), and GMPs. CMPs are multipotent progenitors for both MEP and GMP, but GMPs have lost the ability to develop into red blood cells, and are restricted to generating granulocyte and monocyte cell types. 

New techniques in flow cytometry, mass cytometry, and single-cell RNA sequencing (scRNA-Seq) have led to the identification of markers that define different subsets of hematopoietic progenitors, and studies in mice have suggested that subsets of biomarker-defined GMPs are already directed towards developing into either monocytes or neutrophils. However, as the team notes, “several gaps in understanding the complete neutrophil-lineage hierarchy from CMP to mature neutrophils remain … In humans, the search for a unipotent neutrophil progenitor (hNeP) has been ongoing for decades.” In effect, what scientists haven’t yet identified is the “missing link” of NePs that are formed downstream of GMP, but upstream of the short-term neutrophil precursors. “Over the years, people identified different white blood cell progenitors but the one that was missing was the neutrophil progenitor because we didn't have the tools to pull the populations apart,” says Yanfang Peipei Zhu, Ph.D., postdoctoral fellow at La Jolla Institute for Allergy and Immunology and the study’s first author.

Subsets of immune cells are characterized by the differential expression of cell surface markers. The team’s hunt for the elusive NePs started by exploiting a mass cytometry technique known as cytometry by time-of-flight (CyTOF) to analyze more than 39 biomarkers that define different types of hematopoietic stem and progenitor cells, transient myeloid precursors, and terminally differentiated myeloid cells, including granulocytes, a subset of immune cells that includes neutrophils. Studies using CyTOF and subsequent sc-RNA-Seq analyses of mouse bone marrow cells highlighted a previously unidentified progenitor population — which the researchers labeled #C1 — that demonstrated neutrophil characteristics.

The team then isolated this population of cells from mice and carried out adoptive cell transfer experiments, which confirmed that the cells did differentiate into neutrophils in recipient animals. “Thus, by using high-dimensional mass cytometry and sc-RNA-seq approaches, as well as adoptive transfers in vivo, we have discovered an early-stage committed unipotent NeP (#C1, termed NeP) in mouse BM.”

Interestingly, the researchers found increased numbers of #C1 NePs in the bone marrow and periphery of tumor-bearing mice, “suggesting that there is increased production and egress of these NePs from BM to periphery in response to the tumor microenvironment,” they write. There was also evidence that the progenitors promoted tumor formation, as adoptive transfer of the NePs into mice that subsequently received injections of melanoma tumor cells was associated with increased tumor growth. The #C1 cells were shown to infiltrate the tumor, and about 30% of the #C1 NeP-derived cells expressed PD-L1, an inhibitory costimulatory molecule that contributes to tumor suppression. “Thus, #C1 NeP progenitors respond to melanoma tumor cues and have tumor-promoting functions by producing immune-suppressive progenies,” the authors state.

Given the finding of an association between NePs and tumor growth in mice, the team then identified and isolated candidate NePs from human bone marrow, which they confirmed using adoptive transfer into immunodeficient mice, were neutrophil-specific progenitors. Similar to results in mice, hNePs were found in much higher numbers in the blood of recently diagnosed human melanoma patients than in the blood of healthy volunteers. “This increase of hNeP cells in human melanoma patient blood is consistent with what we have observed for mouse NeP in our mouse melanoma model, suggesting that the hNeP could serve as a biomarker candidate for early cancer detection,” the researchers suggest. Adoptive transfer experiments in mice also indicated that, like the mouse NePs, the human NePs had pro-tumoral activity and mediated solid tumor growth.

Further analyses suggested that compared with mature neutrophils, hNePs may have immunosuppressive properties and promote tumorigenesis by blocking T cell activation. “It seems this progenitor works through T cells, possibly suppressing them and turning them off but we still need to look at the mechanism,” says Dr. Zhu. “It could be one of the factors that have an impact current immunotherapy by making them less efficient.”

The overall findings present new opportunities for both cancer detection and the treatment of neutrophil-related disorders, the researchers suggest. Cancer chemotherapy commonly causes a dramatic reduction in neutrophil levels — a condition known as neutropenia — which makes patients susceptible to potentially dangerous infections. Finding the population of neutrophil progenitors could help scientists identify new approaches to preventing neutropenia in chemotherapy patients.

“Targeting hNeP could rescue patients from undesirable neutropenia,” the authors write. The discovery that hNePs promote tumor growth could also feasibly help scientists to find new therapeutic targets. “… we suggest the possibility that this hNeP itself could be an immune-oncology target, which opens a new field of therapeutic discovery,” they state.

“For many years, the cancer immunology field has been really focused on T cells, which led to the development of checkpoint blockade and CAR-T therapies but there's a whole other arm of the immune system that plays a role in tumorigenesis,” says Dr. Hedrick. “Now, we can study disease where neutrophils execute unique and important functions and investigate further how certain subsets of them promote tumor growth,” Dr. Zhu adds.

 








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