Neutrophils disguise tumors by synthesizing and secreting arginase

From the Houghton lab and collaborating Randolph lab, Clinical Research and Public Health Sciences Divisions

Although the cloak of invisibility only exists in fictional stories, solid tumors have generated a tumor microenvironment (TME) that simulates a biological cloak of invisibility in which tumor cells exist without detection and destruction by immune cells. This biological camouflage has caused significant challenges to the field of immunotherapy, the use of activating the immune cells to target and kill cancer cells within the body. One possible mechanism of tumor cloaking activity may occur via cells in the TME that suppress immune cell proliferation or limit cell viability. For example, Arginase 1 (ARG1) secreted from cells downregulates receptors on immune cells to limit cell activation and proliferation in solid tumors. However, it is unclear which cells are the source of this secreted immune modulator. In mice, expression of ARG1 is the prototypical marker for M2 macrophage. Yet in humans, neutrophils typically synthesize ARG1 when residing in the bone marrow, and there remains no evidence of ARG1 expression by human macrophages. The Randolph and Houghton labs sought to identify the source of ARG1 in non-small cell lung cancer (NSCLC) and determine if immune cells can also synthesize ARG1 while residing in the tumor microenvironment. Their findings were published recently in the Journal of Clinical Investigation and provide insight into features of the TME that disguise tumor cells and participate in dampening the potency of immunotherapy approaches.

“There has been confusion as to whether or not macrophages can express ARG1 in humans,” stated Dr. Houghton. And if not macrophages, then what cells are the source of ARG1? From the myeloid lineages, the researchers analyzed macrophages and neutrophils for expression of ARG1 from NSCLC samples. Negligible ARG1 expression was detected in macrophage yet, a striking ~75% of ARG1 positive cells were neutrophils. Neutrophils typically produce ARG1 while these cells are in the bone marrow and then store ARG1 in granules. For the tumor-associated neutrophils (TANs), the researchers predicted that ARG1 stores would be depleted over time and synthesis of new ARG1 would be needed for continued cloaking activity. Some evidence suggests that TANs can transcribe ARG1 in response to inflammation that occurs outside the bone marrow. Intriguingly, the researchers discovered that 25-40% of TANs actively transcribe ARG1 and cells expressing ARG1 were more likely to have negligible ARG1 protein. These findings suggest a replenishing function of ARG1 in TANs and indicate that the TME, similar to the bone marrow site, is suitable to stimulate ARG1 expression.

Human NSCLC tumor tissue slides were stained for markers of myeloid cell lineages including neutrophils (CD66b and MPO) and macrophage (CD68, CD14 and CD163) and co-stained for ARG1 protein. Neutrophils have the highest abundance of ARG1 positive cells and ARG1 mRNA (red) and protein (yellow) are present in these cells (neutrophils are CD66b+ (green) and/or MPO+ (blue)).
Human NSCLC tumor tissue slides were stained for markers of myeloid cell lineages including neutrophils (CD66b and MPO) and macrophage (CD68, CD14 and CD163) and co-stained for ARG1 protein. Neutrophils have the highest abundance of ARG1 positive cells and ARG1 mRNA (red) and protein (yellow) are present in these cells (neutrophils are CD66b+ (green) and/or MPO+ (blue)). Image modified from publication

To investigate how ARG1 transcription was being activated, the researchers turned to mouse models of lung cancer. In two mouse models, they found similar results as in the human lung cancer tissue samples – TANs had the largest fraction of ARG1 positive cells and the ARG1 secreted from these cells was immunosuppressive. Additionally, co-culture of these mouse TANs with other neutrophils could activate immunosuppressive functions in these other neutrophils, suggesting that the mechanism of increased ARG1 expression relies on a secreted factor present in the tumor microenvironment. ANXA2 was shown previously to act as a ligand for toll-like receptor 2 (TLR2) and TLR4, which leads to the activation of ARG1 expression. To take an unbiased approach in identifying the secreted factor, the researchers performed fast protein liquid chromatography to separate proteins into fractions and then tested each fraction for stimulation of ARG1 expression in neutrophils. Mass spectrometry was used to identify proteins in the active fraction. Intriguingly, ANXA2 was found in the active protein lysate fraction and was confirmed by restricted or enhanced ARG1 expression in neutrophils following ANXA2 inhibition or stimulation, respectively. The researchers also observed that ANXA2 from the tumor cell lysate was insufficient to induce ARG1 transcription in mouse neutrophils lacking TLR2 or Myd88, highlighting a requirement for these signaling factors. They also confirmed that human NSCLC lysates and TLR2 agonists, IL-4 and lipotechoic acid, could activate ARG1 expression in neutrophils isolated from human blood samples.

“The major source of ARG1 within the TME is the neutrophil” and these cells “actually transcribe new ARG1,” stated Dr. Houghton. “This had not been previously described. It was assumed that neutrophils released the ARG1 that they had in granules and that was it,” but “this report shows that neutrophils can replenish the [ARG1] enzyme” for continued immunosuppression. Their investigation of ARG1 regulation led to the discovery of a “novel mechanism by which neutrophils express ARG1 [and this process] involves AnnexinA2, secreted from tumor [cells], to bind TLR2 on neutrophils and produce ARG1 via the MYD88-dependent pathway.”

Some of these findings are still puzzling. For example, “macrophages possess the TLR2/MYD88 pathway but do not make ARG1 in response to AnnexinA2 or other TLR2 ligands the way that neutrophils do,” stated Houghton. “Again, macrophages have the IL-4 receptor but still don’t make ARG1.” In the future, the Houghton lab would like to tease out why macrophage and neutrophils follow different rules for ARG1 regulation.

The spotlighted research was funded by the Seattle Translational Tumor Research program, National Institutes of Health, and a European Commission grant.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Tim Randolph and McGarry Houghton contributed to this work.

Zhang H, Zhu X, Friesen TJ, Kwak JW, Pisarenko T, Mekvanich S, Velasco MA, Randolph TW, Kargl J, Houghton AM. 2022. Annexin A2/TLR2/MYD88 pathway induces arginase 1 expression in tumor-associated neutrophils. J Clin Invest. 132(22):e153643.