Renowned as first responders to threatening infections, neutrophils also happen to feature prominently in the microenvironment of tumors, where they and other immune cells play opposing and frequently mutable roles in promoting—or resisting—cancer progression. Though they’ve been linked to the growth of multiple cancers, including those of the lung and breast, neutrophils can assume multiple functional states, only some of which have such an effect. Identifying those states has, for technical reasons, proved to be quite a challenge.

Now researchers led by Ludwig Lausanne’s Mikaël Pittet have discovered a gene expression program executed by tumor-associated neutrophils (TANs) and a corresponding biomarker that uniformly support cancer cell survival and tumor progression across human and murine tumors. The findings, detailed in the current issue of Cancer Cell, identify TANs characterized by this conserved genetic program as a central variable of the tumor microenvironment (TME) linked to cancer progression. The researchers also describe how the associated marker—a protein called CCL3—functionally supports cancer growth.

“We found that tumors induce in neutrophils a genetic program that sets them on a trajectory of continuous maturation, culminating in a terminal ‘aged’ state characterized by high CCL3 expression,” said Pittet. “These senescent, ‘CCL3^hi’ neutrophils preferentially occupy niches in the TME that are starved of oxygen, engage genetic subroutines that help them adapt to the harsh conditions there and then activate a broad suite of genes that promote the growth and survival of tumors.”

Pittet and his colleagues report that the gene expression of these aged TANs aligns with previous findings linking neutrophils to tumor growth. Senescent TANs, for example, have been found to fuel prostate cancer.

“This conserved pro-tumor state in TANs represents a likely biomarker for predicting patient prognoses across multiple cancer types,” Pittet added.

Although neutrophils are abundant in tumors, the technologies employed to parse cellular states fall short when applied to these cells. This is because the technology—single-cell RNA sequencing (scRNAseq), depends on reading the transcripts of genes in individual cells. Owing to some biological quirk, neutrophils tend to harbor extremely low levels of such RNA transcripts.

Pittet and his team developed a computational method—a probability classifier—to sort neutrophils in distinct functional states based on raw sequencing data and applied it to more than 190 human and murine tumors. These studies, which included the retrospective analysis of existing datasets, uncovered the terminal CCL3^hi state assumed by TANs. Such TANs were seen across the entire spectrum of tumor types examined.

But what exactly does CCL3 do? The researchers show that CCL3 produced by TANs engages a receptor on their surface (CCR1) to transmit signals that drive TANs toward the terminally aged state, bolster their survival in the hypoxic microenvironment and switch on the gene expression programs that drive tumor growth. Accordingly, mice lacking CCL3 in their neutrophils as well as mice whose neutrophils lacked CCR1 failed to support tumor growth, demonstrating that loss of either component of this signaling axis yields the same impairment in TAN-mediated tumor support.

“Our work establishes that tumors actively maintain pro-tumor neutrophils through CCL3 and identifies these cells as a conserved and clinically relevant compartment of cells in the TME,” said Pittet. “This suggests that the genetic and biochemical circuits that ensure the survival of CCL3^hi TANs might be targeted for cancer treatment.”

The findings complement a discovery reported by Pittet and his colleagues in Science in 2023 that the ratio of a pair of genes (CXCL9 and SPP1) expressed by macrophages—related immune cells found in tumors—broadly predicts outcomes for cancer patients. He and his colleagues showed that the genes are linked to a vast network of gene expression programs engaged by multiple cell types in the TME that control the progression of human cancers. When the ratio of CXCL9 to SPP1 is high, gene expression programs in other TME cells indicate a generally anti-tumor slant; a low CXCL9-to-SPP1 ratio, on the other hand, invariably accompanies pro-tumor gene expression signatures across the TME.

The current study suggests that CCL3^hi TANs could be a second variable of similarly singular prognostic significance across tumor types and species.

Source – Ludwig Institute for Cancer Research