Cancer associated fibroblast–T cell crosstalk promotes purinergic synthesis in non-small cell lung cancer (NSCLC)

Sep 22, 2026

Cancer-associated fibroblasts (CAFs) suppress cytotoxic T cell responses in solid tumors by shaping extracellular ATP metabolism in the tumor microenvironment. CD39 and CD73 deplete ATP to generate adenosine, enabling immune evasion. The study investigates CAF–T cell crosstalk in the adenosine pathway in NSCLC, combining flow-sorting, RNA-Seq (nCounter/NanoString), flow cytometry, HPLC measurements, and TCGA/GeoMx analyses to link CD39/CD73 induction with AMP and adenosine production and clinical outcome.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages18

Reading Time45 min

AccessFree to download

Abstract

Introduction

Immune checkpoint inhibition and resistance

CAFs and adenosine-mediated immunosuppression

Methods and Analyses (as described in abstract)

Co-culture and cell sorting

RNA-Seq and flow cytometry

AMP/adenosine measurement by HPLC

TCGA and GeoMx spatial transcriptomics

Key Findings (as described in abstract)

CD4+ and CD8+ T cell upregulation of CD39 and CD73

Increased AMP and adenosine synthesis in vitro

Adenosine signaling linked to poor outcome and gene downregulation

RESEARCH ARTICLE Cancer associated fibroblast–T cell crosstalk promotes purinergic synthesis in non-small cell lung cancer (NSCLC) Lilian Koppensteinera, Charles Locheniea, Richard A. O'Connora, Layla Mathiesona, Liam Neilsona and Ahsan R. Akrama, b aCentre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK; bCancer Research UK Scotland Centre, Institute of Genetics and Cancer, The University of Edinburgh, Edinburgh, UK ABSTRACT The most abundant stromal cells of the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs), inhibit the cytotoxic T cell response in solid tumors. CD39 and CD73 together deplete extracellular ATP in the TME to produce adenosine, which can promote tumor immune evasion. We aimed to investigate the role of CAF-T-cell crosstalk in the adenosine pathway in NSCLC. CD4+ and CD8+ T cells were flow sorted from in vitro culture with or without CAFs, and RNA-Seq was performed using the nCounter platform (NanoString). The expression of cell surface markers involved in adenosine production was assessed using flow cytometry in CAFs and peripheral T cells from healthy donors or early NSCLC patients following in vitro co-culture. Purinergic synthesis of AMP and adenosine was measured by HPLC. TCGA data was used to investigate the translational relevance of adenosine signaling in NSCLC, and NanoString Geomx was performed to investigate differences in the transcriptomics of tumor stromas with a high versus low adenosine signature. CD4+ and CD8+ T cells isolated from in vitro co-culture with CAFs show upregulated expression of CD39 and CD73, which results in functionally relevant production of AMP and adenosine. TCGA data illustrates that adenosine signaling is predictive of poor outcome in lung squamous cell carcinoma. Spatial transcriptomics (GeoMx) of the tumorstroma of early untreated NSCLC patients shows a downregulation of immune-related genes in lung squamous-cell carcinoma. CAF T cell crosstalk promotes the expression of CD39 and CD73 on CAFs and T cells, resulting in an increased synthesis of AMP and adenosine in vitro. ARTICLE HISTORY Received 22 February 2026 Revised 16 July 2026 Accepted 17 July 2026 KEYWORDS Cancer-associated fibroblasts; tumor microenvironment; TILs; NSCLC; adenosine Introduction Immune checkpoint inhibition (ICI) has revolutionized cancer care, although disappointingly, the majority of non-small cell lung cancer (NSCLC) patients remain unresponsive or develop acquired resistance in approximately 80% of patients. 1 The tumor microenvironment (TME) of NSCLC harbors multiple immunosuppressive pathways that can prevent a successful T cell response. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells of the tumor microenvironment (TME) and play a crucial role in modulating the immune landscape via multiple mechanisms. This includes inhibition of the T cell response by reducing T cell proliferation, promoting T cell exhaustion, apoptosis and differentiation into regulatory phenotypes (reviewed) .2 In solid tumors, CAFs have been shown to express ectonucleotides involved in the production of the immunosuppressive signaling nucleoside adenosine, another potent immune checkpoint.3-5 Adenosine canelicit broad physiologic responses from immune and non-immune cells and negatively impact the immune response to cancer. It signals via type 1 purinergic receptors (A1, A2A, A2B, A3), resulting in effects opposite to the pro-inflammatory effects of ATP. The adenosinergic receptors A2AR and A2BR are upregulated by inflammatory mediators such as TNF-α as a negative feedback mechanism to dampen an exaggerated response.6 The TME of solid tumors displays high concentrations of adenosine and co-opts CONTACT Ahsan R. Akram [Ahsan.Akram@ed.ac.uk](Ahsan.Akram@ed.ac.uk) Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK Supplemental d

FAQs

What is the main immunosuppressive mechanism discussed for NSCLC?

CAFs contribute to adenosine generation in the tumor microenvironment by CD39/CD73-mediated depletion of extracellular ATP, promoting T cell immune evasion.

How were the CAF–T cell interactions experimentally studied?

CD4+ and CD8+ T cells were flow sorted from in vitro cultures with or without CAFs, followed by RNA-Seq (nCounter/NanoString) and flow cytometry assessment of adenosine-pathway markers.

What did the study report about CD39 and CD73 and downstream purinergic synthesis?

T cells co-cultured with CAFs showed upregulated CD39 and CD73, leading to functionally relevant increased production of AMP and adenosine; HPLC was used to measure AMP/adenosine.

Sep 22, 2026

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