Efferocytosis in Cancer: How a New Tool (Effero-seq) Reveals That Tumor-Associated Macrophages Are Reprogrammed by the Dying Cells They Eat

A new forensic tool for the body's quietest crimes

Apoptosis and Efferocytosis

Every day, vast numbers of cells in our body die through controlled self-destruction called apoptosis. Apoptosis helps with tissue renewal and remodeling. Clearing away the bodies falls to macrophages, which are the immune system's cleanup crew. They do this through a process called efferocytosis, by which they engulf and digest dying cells. During efferocytosis, macrophages release anti-inflammatory signals to keep the tissue calm.

Open question

Does eating a dying cell change the macrophage that ate it? If it does, how long does this change last?

Why is this relevant to health?

Efferocytosis also occurs in disease. Previous studies have correlated efferocytosis to diseases like atherosclerosis, neurodegeneration, and even cancer. However, they have so far been just correlations. Whether efferocytosis simply marks diseased tissue or actively drives disease has been unknown.

That gap is what a team from the Cohen Lab at Tel Aviv University set out to close. Are macrophages that eat dying cells innocent bystanders, or active participants?

They began by stepping back to answer a simpler question: when a macrophage eats a normal, healthy apoptotic cell, does the act leave a lasting mark?

How do you track a cell that eats the evidence?

To follow efferocytosis, you first need to know which macrophages have eaten. The clever idea: tag the dying cell with a label that remains dark at normal pH but fluoresces in the acidic interior of the lysosome. This ensures that the tag only glows once the cell has been engulfed and digestion begins.

While this may look simple in principle, the experimental challenges to set this up are many. Firstly, efferocytosis is fast and uneven and can take anywhere from a  few hours to days to complete. Secondly, a single macrophage can eat several dying cells in succession. Thirdly, the apoptotic cell is ultimately digested and destroyed, making it challenging to compare efferocytosing macrophages analyzed at different time points. Finally, fluorescent labels that are not chemically conjugated to the apoptotic cargo can leak, leading to mislabeled cells.  

To fix these issues, the fluorescent tag needs two main features.

1) It has to be covalently bonded to the apoptotic cargo to prevent leaking.

2) The tag has to preferably be membrane impermeable to ensure it remains in the lysosome intact for hours.

These features will ensure that you can properly mark an efferocytosing macrophage even after the apoptotic cell it ate is digested.

Enter pHrodo.

pHrodo is nearly non-fluorescent at neutral pH when it is present on the cell surface and in the cytoplasm. However, it becomes brightly fluorescent as the pH drops within the acidifying phagosome and lysosome. The researchers covalently attached it to apoptotic cells. Furthermore, pHrodo remains within the lysosomes owing to its membrane impermeability. The resistance to photobleaching is an added bonus that enables tracking cells over a longer time period.

In the dish

The team chemically triggered apoptosis in thymocytes and labeled them with pHrodo. This was then fed to macrophages in culture. As expected, macrophages that ingested the labeled cargo via efferocytosis glowed, whereas others did not. Moreover, the tag's brightness correlated with progress in the efferocytosis process.

Single-cell sequencing comparing pHrodo+ and pHrodo- macrophages revealed more insights into their gene activity. By 24 hours, the efferocytosing macrophages had downregulated MHC genes critical for antigen presentation to the immune system while upregulating others.

In the animal

The team delivered pHrodo-labeled apoptotic cells into the noses of mice. Alveolar macrophages are major efferocytosing cells in the lung that also dialed down their MHC genes, similar to the in vitro experiment. These cells upregulated other genes involved in lysosomal activity, target recognition, and immune-modulating programs.

The common thread

Efferocytosing macrophages consistently suppressed their MHC genes in both the in vitro and in vivo models. Additionally, pHrodo brightness correlated with the extent of efferocytosis.

The effero-score

Just as the pHrodo brightness increased as efferocytosis progressed, researchers also identified other genes whose expression increased in parallel with the fluorescence signal. They clubbed 66 such genes and defined an effero-score as a molecular fingerprint of a macrophage that performed efferocytosis.  

Making sure the score is specific to efferocytosis

Macrophages ingest many substances via phagocytosis and pinocytosis, including bacteria, yeast, and other debris. It was important at this stage to differentiate efferoscore from these secondary processes. To do this, the team used pHrodo-labeled E.coli and zymosan (a yeast-derived particle) as cargoes to trigger general phagocytosis. They found that macrophages engulfing apoptotic cells exhibited a distinct signature different from that of those involved in clearing bacteria or fungi.

Does it show up in disease?

With a specific signature (effero-score) in hand, the team asked whether efferocytosing macrophages appear in disease. Rather than run new experiments, they reanalyzed published single-cell datasets from other labs, applying the effero-score as a lens. They observed that distinct macrophage subsets showed higher effero-score across a range of conditions:

Alzheimer's and ALS: microglia

Lung fibrosis: interstitial macrophages

Breast cancer: perivascular macrophages

Non-small-cell lung cancer: interstitial macrophages

This confirmed the presence of efferocytosing macrophages in disease tissue. However this still did not answer whether efferocytosis is driving disease states or leave macrophages as bystanders.

Bystander or driver? The tumor test

To better understand the role of efferocytosing macrophages in disease, the team used a melanoma model. Apoptotic cargo containing pHrodo-labeled B16 melanoma cells was co-injected with unlabeled tumor cells into mice. A control group received live tumor cells without any labeled apoptotic cargo.

Because tumors contribute to cell death on their own, efferocytosis occurs naturally within them. Therefore, although the control group would not produce pHrodo+/- macrophages, it would still have subsets bearing the effero score signature.

In the labeled group, pHrodo-positive macrophages were indeed present in the tumor. Similar to previous observations, they were distinct from antigen-presenting subsets. They had a high effero-score along with other genes that followed a similar expression pattern. This shows that the effero-score is a reliable tool for tracking efferocytosis and can be adapted to specific disease contexts.

As expected, effero-score allowed researchers to identify efferocytosing macrophages in the control group. This proved that effero-score can identify efferocytosing cells in datasets where no labels were ever used, making it a valuable resource for better understanding human disease.

Does it matter for patients?

To test clinical relevance, the team analyzed single-cell data from people with uveal melanoma, a rare cancer of the eye. Macrophage subsets with a high effero-score also expressed genes that promote blood vessel growth, such as VEGFA. And patients whose tumors showed higher expression of these genes had worse overall survival.

What's actually changing inside these macrophages?

To find out whether macrophages were reprogrammed, the team used ATAC sequencing to map DNA accessibility. They observed that pHrodo-positive macrophages from the mouse tumors had less accessible IFN-gamma signaling. Moreover, this loss of IFN-gamma responsiveness persisted even outside the tumor, suggesting the reprogramming is stable. Since IFN-gamma is a key driver of anti-tumor immunity, this meant the macrophages had been reprogrammed to blunt their own anti-tumor response.

Two findings come together

So the tumor-associated efferocytosing macrophages were doing two things at once:

1. Blunting their response to IFN-gamma and weakening anti-tumor immunity.

2. Switching on genes that promote blood-vessel growth.

To gain more clarity, the team then used spatial transcriptomics to map efferocytosing macrophages and blood vessels in the tumor microenvironment (TME). pHrodo+ macrophages clustered close to blood vessels in the TME.

The takeaway

Efferocytosing macrophages are reprogrammed. In tumors, reprogramming leaves macrophages ineffective at anti-tumor responses. They also promote blood vessel growth near them, facilitating tumor growth.

Why this matters

Until now, efferocytosing macrophages have been hard to track because the process is rapid and there has been no clean way to design the experiment. This study shows how the modern toolkit with single-cell sequencing, ATAC-seq, and spatial transcriptomics helped crack that puzzle. The result is a new forensic tool for watching what happens after a macrophage eats a dying cell, and the effero-score as a fingerprint that can be used to study complex diseases.

It also establishes the long-assumed link between efferocytosis and its pro-tumor function. The study reveals a second route by which these cells help tumors: not only standing down from the fight, but building the vasculature that nourishes the tumor. For cancer biologists, that's a new target for therapies.

But is it only cancer?

Apoptosis happens every day, mostly in the quiet service of renewing our tissues. This study examined macrophage reprogramming only in the context of cancer.

But does it stop there? Could the same reprogramming occur in the presence of a vaccine like BCG, which is known to reprogram immune cells such as macrophages by trained immunity? Ageing is another avenue with more cell death and less renewal- what could efferocytosing macrophages teach us about aging and susceptibility to diseases?