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Rescuing Memory T Cell Chemotaxis to Boost Tumor Infiltration

By knocking down the adenosine A2A receptor with silencing mRNA inside a lipid nanoparticle, researchers restore chemotaxis in head and neck cancer memory T cells.

Adenosine, as we all know, shows up in a lot of places and has a lot of different roles to play. One of those happens to be in the tumor microenvironment (TME), where it accumulates and suppresses important T-cell behavior like chemotaxis. Adenosine in the TME binds to the adenosine A2A receptor (A2AR) and triggers a signaling cascade that eventually inhibits the Ca2+-dependent channel, KCa3.1, which controls human T-cell migration.

This is a major problem for the body’s ability to successfully deal with tumors since that process relies in part on a high degree of CD8+ T cell infiltration. If those cytotoxic memory T cells are unable to migrate effectively, tumor infiltration is low and the response to treatments like immunotherapy can be poor. In the case of solid tumors, such as head and neck squamous cell carcinoma (HNSCC), circulating CD8+ T cells seem to show a higher sensitivity to adenosine, which severely limits their ability to infiltrate the TME.

Could targeting the adenosine pathway be a solution?

All Eyes on A2A Receptors

Dr. Hannah Newton and a team from the University of Cincinnati College of Medicine set out to determine exactly that.1 An existing body of work already sets the precedent for the A2AR as a valid target to increase cytotoxic T-cell function and even reduce tumor burden. The adenosine pathway, therefore, looks to be an excellent target to improve T-cell migration and ultimately boost the response the checkpoint inhibitors and T-cell therapies.

The issue here is that A2AR expression isn’t limited to the TME: it’s on multiple immune cells across multiple tissues. We also know that the adenosine pathway is involved in a range of biological processes, from angiogenesis to regulation of motor activity and function. 

Rather than a systemic approach – and the toxicity that goes alongside systemic pharmacological A2AR inhibitors – a much more targeted method would be needed to affect A2ARs specifically on memory T cells.

Targeting Cells With Fatty Nanoparticles

Take a ball of lipids, insert whatever cargo you want to deliver, engineer an additional component to target specific cell types, and you’ve got yourself a lipid nanoparticle (NP). Admittedly it’s a little more complex than that, but the result is essentially vesicles that target specific cells, are taken up by endocytosis, and can deliver small molecules like silencing/interfering RNA directly into the cell. 

In this case, Dr. Newton and team assembled a ball of phospholipids, into which they inserted siRNA against ADORA2A (the gene encoding the A2AR). To make sure the lipid NPs reached the right T cells, NPs were labeled with fluorescent streptavidin and biotinylated targeting antibodies – either anti-CD45RO or anti-CD8 since CD8+CD45RO+ T cells make up 90% of the T-cell population in solid tumors.

Their proof-of-concept experiments on cultured HNSCC patient cells showed that while CD8- and CD45RO-labelled NPs were specific for their respective cell types, CD8-labelled NPs didn’t knockdown ADORA2A expression. This was because cells quickly internalized the CD8-labelled NPs into the lysosome, effectively destroying the siRNA. CD45RO-labeled NPs, on the other hand, were much better at escaping the lysosome and successfully released ADORA2A siRNA, knocking down ADORA2A expression.

To show the extent of A2AR knockdown with their lipid NPs, researchers used our Anti-Human Adenosine A2A Receptor (extracellular) Antibody (#AAR-007) and Human Adenosine A2A Receptor (extracellular) Blocking Peptide (#BLP-AR007) with flow cytometry. The specificity of the antibody was confirmed by using the blocking peptide in a parallel flow cytometry assay and by measuring mRNA levels of the targeted A2AR.

Rescuing chemotaxis

Adenosine accumulation at the TME reduces memory T cell chemotaxis by as much as 80% in HNSCC patients. The question now was could nanoparticles chemotaxis in these T cells?

To test this, the researchers incubated activated CD8+ memory T cells from HNSCC patients with CD45RO-labelled NPs for 72–96 hours. The cells were then resuspended in a collagen matrix to more accurately recreate the 3D environment of the TME. To induce chemotaxis, a chemokine gradient was created with CXCL10 alone, or CXCL10 plus adenosine.

The results were impressive, and the chemotaxis inhibitory effects of adenosine were significantly reduced by the CD45RO-labeled NPs delivering their cargo of ADORA2A siRNA. It was shown how t-cell sensitivity to adenosine was reduced and their ability to migrate effectively in an adenosine-rich, immunosuppressive environment was restored.

The work here from Dr. Newton et al. shows a novel approach to target the adenosine pathway and promote T-cell infiltration into the TME. We’ll certainly be keeping an eye on this research to see the outcome of this approach in in vivo models.

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Reference

  1. Newton, H. S., Chimote, A. A., Arnold, M. J., Wise-Draper, T. M. & Conforti, L. Targeted knockdown of the adenosine A2A receptor by lipid NPs rescues the chemotaxis of head and neck cancer memory T cells. Mol. Ther. – Methods Clin. Dev. 21, 133–143 (2021).

Photo by National Cancer Institute.