Biology subjects

Padget, J.

Publications and source records attributed to Padget, J..

4 recordsLinked to original sources

bioRxiv2026

Integrated Spatial Multi-omic Profiling Identifies HSV-associated Inflammatory Macrophage Niches Linked to Oncolytic Virotherapy Response in Melanoma

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

cancer biology↗

bioRxiv2025

AXL tyrosine kinase inhibition rescues immune checkpoint blockade-resistant melanoma in a tumor microenvironment-dependent fashion

BackgroundImmune checkpoint blockade (ICB) achieves durable responses in approximately half of patients with advanced melanoma, but the mechanistic basis for resistance in the remaining patients remains incompletely defined. AXL tyrosine kinase has emerged as a candidate resistance mediator, yet clinical development of AXL inhibitors has yielded heterogeneous results in unselected patient populations, suggesting that cell-type and tumor microenvironment context may critically govern therapeutic response. MethodsWe characterized AXL expression across cell types in ICB-resistant melanoma using publicly available single-cell RNA sequencing datasets and The Cancer Genome Atlas (TCGA). In vivo efficacy was assessed in the YUMM1.7 PD-1-resistant syngeneic melanoma model using three pharmacologically distinct AXL inhibitors (warfarin, bemcentinib, and cabozantinib) as monotherapy and in combination with anti-PD-1 therapy. Tumor-associated macrophage (TAM) context-dependency was established using anti-CSF1R and anti-F4/80 depletion strategies. Functional PD-L1:PD-1 checkpoint interactions were quantified in tumor sections by immune Forster Resonance Energy Transfer (iFRET). TAM secretome reprogramming was characterized by 40-plex Luminex immunoassay in polarized RAW264.7 macrophages. ResultsAXL expression in ICB-resistant melanoma was predominantly localized to TAMs rather than tumor cells by single-cell analysis, with AXL+ TAMs distributed across both M1-like and M2-like phenotypic compartments. AXL inhibition significantly reduced tumor burden and synergized with anti-PD-1 therapy in vivo; however, efficacy was abolished by depletion of the monocyte-derived myeloid compartment (anti-CSF1R) and enhanced by depletion of tissue-resident TAMs (anti-F4/80), establishing TAM-context dependency. iFRET revealed a paradoxical gain of PD-L1:PD-1 interaction efficiency in anti-PD-1-treated resistant tumors, a functional resistance signature detectable by iFRET but not by PD-L1 expression that was reversed by AXL combination therapy. In vitro secretome profiling demonstrated that combination therapy reprograms macrophage secretome in a polarization-context-dependent manner, amplifying pro-inflammatory cytokines including IL-6 and GM-CSF in M2-like macrophages while selectively dampening T cell chemokine production. ConclusionsThese findings establish AXL as a TAM-resident immune target in ICB-resistant melanoma whose therapeutic relevance is governed by tumor-immune micronenvironment (TiME) macrophage composition rather than tumor cell AXL expression. TAM polarization contexture represents a candidate stratification axis for AXL inhibitor-based combination strategies, and iFRET-measured checkpoint interaction offers a functional complement to PD-L1 expression for monitoring resistance and response. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSO_LIAXL tyrosine kinase has been studied predominantly as a tumor-intrinsic mesenchymal marker and therapeutic target in melanoma, with prior clinical development of AXL inhibitors predicated on tumor cell AXL expression as the primary biomarker; the contribution of TAM-expressed AXL to ICB resistance and the dependence of therapeutic response on TiME composition have not been defined. C_LI What this study addsO_LIAXL expression in ICB-resistant melanoma is predominantly localized to tumor-associated macrophages distributed across both M1-like and M2-like phenotypic compartments, reframing AXL as a TAM immune target whose therapeutic relevance is governed by TiME macrophage composition rather than tumor cell AXL expression. C_LIO_LICombination AXL inhibition and anti-PD-1 reprograms the macrophage secretome in a polarization-context-dependent manner and reverses a paradoxical gain of functional PD-L1:PD-1 checkpoint interaction in resistant tumors -- a resistance signature detectable by spatial iFRET but not by PD-L1 expression. C_LI How this study might affect research, practice or policyO_LIThe context-dependent effects of AXL inhibition (immunostimulatory in M2-heavy TiMEs, potentially counterproductive in M1-heavy TiMEs) indicate that TAM polarization profiling should be incorporated into clinical trial design for AXL inhibitor-based combinations, and that unselected enrollment may obscure meaningful efficacy signals in the subset most likely to benefit. C_LIO_LIiFRET-based quantification of functional checkpoint interaction represents a candidate dynamic biomarker for ICB resistance monitoring that is orthogonal to PD-L1 immunohistochemistry and may identify resistant patients who retain immunosuppressive checkpoint interactions that can be disrupted by AXL targeting. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=170 HEIGHT=200 SRC="FIGDIR/small/667666v2_ufig1.gif" ALT="Figure 1"> View larger version (90K): org.highwire.dtl.DTLVardef@521de1org.highwire.dtl.DTLVardef@127a933org.highwire.dtl.DTLVardef@d56132org.highwire.dtl.DTLVardef@e03ef9_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Functional Spatial Mapping of the Tumour Immune Microenvironment In Advanced Melanoma Patients

IntroductionCurrent spatial proteomic approaches quantify immune checkpoint expression but do not directly measure functional receptor/ligand (PD-1/PD-L1) interactions within the tumor immune microenvironment (TiME). Therapeutic antibodies disrupt receptor-ligand interactions and do not target protein abundance. Methods that resolve functional checkpoint interactions provide biologically distinct insight beyond expression-based assays MethodsWe combined computation and quantitative spatial imaging, FuncO:TiME, [Functional Oncology Mapping (FuncOmap)], to map PD-1/PD-L1 interaction states to spatially defined regions of the TiME, in clinically annotated melanoma specimens, collected before and after neoadjuvant immune checkpoint blockade (ICB), ResultsFuncOmap spatially quantified millions of per-pixel PD-1/PD-L1 interactions demonstrated spatial heterogeneity in checkpoint interaction, not reflected by PD-1 expression levels alone. Post-treatment tissues exhibited increased PD-1/PD-L1 interaction states despite no corresponding increase in expression, indicating persistent or augmented functional checkpoint interaction despite therapy. Integration with spatial immune profiling further demonstrated that checkpoint interaction intensity can be contextualized within distinct immune cell populations. ConclusionWe have established the feasibility of spatially resolved functional checkpoint mapping in human melanoma tissues. We demonstrate that receptor-ligand interactions diverge from protein expression patterns. By enabling direct interrogation of functional checkpoint interaction dynamics within intact tissue architecture, FuncO:TiME advances a functional paradigm for studying immune regulation in cancer.

cancer biology↗

bioRxiv2025

AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

RationaleIdiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by aberrant alveolar repair and excessive fibrosis. The TAM-family receptor tyrosine kinase AXL, activated by GAS6 and PROS1, is implicated in tissue remodeling, but ligand-specific AXL signaling during alveolar repair remains poorly defined. ObjectivesTo investigate ligand specific AXL signaling in IPF and how it impacts epithelial proliferation and repair after alveolar injury in-vivo and in-vitro. MethodsSingle cell RNA sequencing was utilized to understand cell specific expression patterns in IPF patients, followed by functional studies in primary human cell culture and functional spatial digital profiling (FuncOmap) analysis in patient tissue. Longitudinal assessment of repair process after alveolar-specific injury in-vivo was used to complement the in-vitro approach. ResultsAXL expression showed enrichment in basal and aberrant basaloid cells of IPF patients. In-vitro GAS6 increased proliferation of basal cells, while PROS1 counteracted this effect. FuncOmap analysis demonstrates direct in-situ interactions between AXL and both ligands, providing evidence for biological relevance. Investigating longitudinal repair processes in-vivo revealed dynamic regulation of AXL ligands as well as AXL. ConclusionsThese findings highlight the importance of ligand-specific AXL signaling in lung repair and suggest that it dysregulation may contribute to IPF pathogenesis, offering potential therapeutic targets for restoring normal repair processes.

molecular biology↗