Biology subjects

Burley, A.

Publications and source records attributed to Burley, A..

5 recordsLinked to original sources

bioRxiv2026

Spatially-resolved multimodal profiling identifies functionally-heterogeneous cancer-associated fibroblasts associated with poor radiotherapy outcomes in muscle-invasive bladder cancer.

Background: Cancer-associated fibroblasts (CAFs) contribute to systemic therapy resistance in muscle-invasive bladder cancer (MIBC), but their functional heterogeneity and relevance to curative bladder-preserving radiotherapy, are poorly understood. Methods: Transcriptomic analysis was performed on 279 tumours from BC2001, a phase 3 radiotherapy clinical trial. To study CAF heterogeneity, we integrated bulk RNA-seq, single-cell spatial analysis of multiplex immunofluorescence images and quantification of extracellular matrix (ECM) features in 155 MIBC biopsies. The functional heterogeneity of distinct CAF populations was evaluated by single-nuclear RNA-seq. Spatial interactions between CAF populations and CD8+ T-cells was assessed and the relevance of lymphocytes to radiation responses was evaluated in a CAF-enriched murine bladder cancer model (BBN963). Results: BC2001 patients with CAF-enriched tumours had worse overall survival (HR=1.671, 95% CI 1.221-2.287, Log-rank p=0.0012). CAF abundance and antigen expression was highly heterogenous. Podoplanin (PDPN) was expressed on the majority of CAFs and was associated with inflammatory pathways. Enrichment of CAF gene signatures was associated with a significant increase in CAFs expressing fibroblast activation protein (FAP) (p=0.004) and dense ECM features (p=0.0004). Fifty-three percent of tumours exhibited stromal CD8+ T-cell exclusion with significant enrichment in FAP-dominant neighbourhoods (p<0.001). In vivo, lymphocytes were critical for radiation-induced tumour control, indicating that immune cold or excluded tumours may have limited radiotherapy responses. Conclusion: In MIBC, CAFs are associated with poor radiotherapy outcomes. Multiple mechanisms are deployed by functionally-heterogeneous CAFs, including promotion of chronic inflammation by PDPN+ CAFs and ECM remodelling by FAP+ CAFs which impact CD8+ T-cell distribution and radiation responses.

cancer biology↗

bioRxiv2026

Development of a multiplex immunofluorescence panel to study heterogenous cancer-associated fibroblast subtypes with spatial resolution

BackgroundSingle cell RNA sequencing provides a wealth of information to explore the complexities of the tumour microenvironment, but crucially the spatial topology of the tumour is lost and studying cellular interactions is limited. Spatial transcriptomics aims to address this however the technique remains cost prohibitive for the generation of data from meaningfully-sized clinical cohorts. In contrast, spatial proteomic profiling with multiplex immunofluorescence, preserves spatial interactions, is relatively cost accessible, and is scalable for large clinical cohorts to address powerful translational questions. Whilst multiplex approaches have advanced in recent years, we note that cancer-associated fibroblasts (CAFs) have been explored in less detail, potentially due to difficulties associated with CAF heterogeneity and the diversity of markers used to define them. MethodsWe designed, optimised, and validated a multiplex immunofluorescence panel that combines four frequently used CAF markers; alpha smooth muscle actin (SMA), fibroblast activation protein (FAP), podoplanin (PDPN) and platelet-derived growth factor receptor alpha (PDGFR) with CD8 and pan-cytokeratin. Here we share our methodology and the practical considerations taken to inform the final panel design. We also highlight the benefits of robust optimisation experiments.

pathology↗

bioRxiv2026

Addition of chemotherapy to radiotherapy promotes progenitor-exhausted CD8⁺ T-cell clonal dominance in head and neck cancer

Concomitant chemoradiotherapy (CRT) is a standard-of-care for unresectable locally-advanced head and neck squamous cell carcinoma (LA-SCCHN), but its immune effects, particularly compared to radiotherapy (RT) alone, remain unclear. Using syngeneic murine models, we integrated Nr4a3-Tocky reporter analysis with single-cell transcriptomics and T-cell receptor clonotyping comprehensively to profile intratumoural CD8 T-cells following RT/CRT. We show that CRT uniquely drives robust antigen-specific clonal expansion and biases differentiation toward progenitor (or precursor) exhausted (TPEX) phenotypes, while RT favours terminal exhaustion (TEX). Single-cell analyses reveal CRT-induced clonal dominance within TPEX subsets, suggesting the potential for enhanced immune reinvigoration. In peripheral blood mononuclear cells (PBMCs) from patients treated with CRT, high levels of TEX cells were found early and at 3 months post-treatment, with delayed peripheral TPEX expansion at 3 months, indicating phased progenitor recovery. These findings demonstrate distinct immunological remodelling by CRT versus RT and underscore the critical importance of treatment timing for optimising combination immunotherapy strategies in LA-SCCHN.

cancer biology↗

bioRxiv2025

Rapid expansion of podoplanin-positive fibroblasts following radiation limits the anti-tumour CD8+ T-cell response to radiotherapy.

Radiotherapy is known to cause changes in the tumour stroma which can undermine treatment efficacy. Our understanding of this process has historically centred around effects driven by Transforming Growth Factor-beta (TGF-{beta}) and alpha-smooth muscle actin (-SMA)+ fibroblasts. Here, we identified a rapid expansion of podoplanin (PDPN)+ fibroblasts following radiotherapy in breast, head and neck and melanoma tumours. This fibrosis was not dependent on TGF-{beta}, but was downstream of a radiotherapy-induced adaptive immune response. CD8+ T-cells entering the tumour after radiation were sequestered at the interface between residual tumour cells and PDPN+ fibroblasts and failed to enter the tumour core. Genetic deletion of PDPN in fibroblasts impacted their cytoskeleton and ability to organise extracellular matrix. This was associated with increased CD8+ T-cell entry and spontaneous tumour regression. Overall, we identify a mechanism whereby PDPN+ fibrosis limits immune-mediated radiation cell kill and demonstrate that disruption of PDPN signalling favours tumour control. SignificanceIn this study we show that rapid podoplanin (PDPN)+ fibroblast expansion following radiotherapy limits immune-mediated radiation cell kill. Targeting PDPN and associated downstream signalling improves tumour control and is a promising strategy in combination with radiotherapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/680949v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1711bb0org.highwire.dtl.DTLVardef@d0c3a1org.highwire.dtl.DTLVardef@1db88adorg.highwire.dtl.DTLVardef@1ea3ab6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Sculpting the tumour microenvironment by combining radiotherapy and ATR inhibition for curative-intent anti-PD-L1- and anti-NKG2A-based adjuvant immunotherapy

Despite some success in other cancer types, the results of combining radiotherapy/chemoradiotherapy and immune checkpoint blockade have been disappointing in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). For such a potentially radiocurable disease, there remains an imperative to explore novel combination approaches. Here, we show that combining ATR inhibition with radiotherapy (ATRi/RT) increases the frequency of highly activated NKG2A/PD-1 double-positive T cells in patients and in animal models of HNSCC. Addition of dual anti-NKG2A and anti-PD-1/-PD-L1 blockade to ATRi/RT in the adjuvant, post-radiotherapy setting induces a robust antitumour immune response in HNSCC preclinical models. Efficacy of the combination regimen relies on CD40/CD40L costimulatory-mediated infiltration of activated/proliferative/memory CD8 and CD4 conventional T cells with persistent or new T cell receptor (TCR) signalling, respectively, as defined by tracking of T cell dynamics. In this favourable therapeutic context, TCR sequencing shows increased richness of the TCR repertoire and the emergence of numerous and large TCR clusters that share antigen specificity in response to full combination therapy. Collectively, our data point towards promising combination approaches for future clinical testing in HNSCC.

immunology↗