Microwave ablation induces heating of tissue. High energy results in thermal cell death, but low energy treatment without tissue necrosis has been shown to induce cutaneous immunity in an HPV wart model. It is approved as an effective cancer treatment for solid organ cancers, and therefore it is of interest to know if microwave delivered directly to the skin holds potential for treatment of skin cancer. This pilot study focused on transcriptomic profiling of cutaneous melanoma metastases to investigate molecular changes associated with microwave therapy. Seven adult patients with skin metastases from malignant cutaneous melanoma, not resolving on standard treatment, were recruited. Microwave energy was applied to separate melanoma metastases. Morphological, histological, and transcriptomic changes assessed via tissue RNA sequencing were evaluated. Three participants showed complete response, while four showed partial response by histological assessment. In complete responders, skin lesion RNA sequencing after treatment, compared with baseline, identified increased inflammation (CXCL5, CXCL8, IL1A, COL1A1) and downregulated cancer markers (PRAME, S100B, MLANA, STK32A). Compared with partial responders, complete responders showed enrichment of FABP4 and reduced expression of cancer markers. Microwave therapy produced local tumor responses and associated inflammatory transcriptomic changes in complete responders, supporting further clinical evaluation in cutaneous melanoma metastases.

Abstract Introduction and aims Malassezia species have been found at higher loads in common skin conditions compared with healthy skin. Whether they drives host responses towards specific cutaneous inflammatory phenotypes or act as an opportunistic microbe at inflammation sites remains unclear. We set out to characterize the keratinocyte transcriptional response to Malassezia globosa (MG) and Malassezia furfur (MF) using a reconstituted human epidermis (RHE) model composed of differentiated keratinocytes, and to identify species-specific host–pathogen interactions. Methods RHE models were stimulated with MG and MF under varied dose and timepoint conditions and sequenced (Illumina NovaSeq6000). Transcriptomic data were analysed using R and checked for quality control (MultiQC), before data analysis (Kallisto, tximport and edgeR) and comparison with healthy or psoriasis lesions (GSE173706). Ontology was assessed using functional enrichment analyses (iDEP, ShinyGO and VissE Cloud) and ingenuity pathway analysis. RHE inflammatory protein expression was also evaluated (Olink Target 48 Cytokine Panel). Results MF-stimulation induced 4052 DEGs compared with 14 458 DEGs following MG-stimulation (P < 0.05, false discovery rate = 0.05). Principal component analysis revealed species-dependent clustering in the keratinocyte response to Malassezia. Both Malassezia species induced barrier-related transcripts and proinflammatory cytokines (CXCL1, CXCL8, CCL20) and upregulated interleukin (IL)36G. MG induced additional cytokines (IL17C, IL23A, TNFAIP6 and IRAK2), demonstrating a 42% overlap with a psoriasis disease network, indicating shared upstream regulators and the upregulation of IL17 and tumour necrosis factor pathway-related molecules. Contrastingly, MF induced lipid metabolism-associated transcripts (CYP4F22). Protein analysis confirmed MG upregulation of key immune mediators (IL1β, IL18, IL6, IL17C, TNF, CCL2, CXCL8 and GM-CSF). MG-induced keratinocyte signatures were recapitulated in psoriatic keratinocyte single-cell RNA-sequencing data, identifying the IL36G–IFNAR2 signalling axis as a link between epidermal and inflammatory responses. Conclusions Malassezia species elicit divergent keratinocyte transcriptomic signatures. MG promotes a psoriasis-like inflammatory profile, while MF favours a barrier-focused response with metabolic adaptation. These findings support the hypothesis that individual Malassezia species may facilitate distinct patterns of cutaneous phenotype.

Abstract Introduction and aims Cutaneous melanoma is highly metastatic and resistant to conventional therapies. Its outcomes are shaped by complex genetic and microenvironmental mechanisms that remain incompletely understood. Previous work showed that microwaves can prime keratinocytes to enhance dendritic cell-mediated immune responses, suggesting potential for modulating antitumour immunity. This study aimed to evaluate microwave therapy in melanoma metastases using spatial transcriptomics to identify molecular and cellular signatures associated with treatment response. Methods Nine participants with stage IV or inoperable stage III melanoma who were unsuitable for alternative treatments received microwave therapy. Treatment effects were assessed at baseline and three weeks post-treatment, and patients were classified as complete or partial responders based on histological evaluation. Punch biopsies pre- and post-treatment were processed as formalin-fixed paraffin-embedded tissue for 10X Visium spatial transcriptomics. Analyses focused on cell type-specific transcriptional changes and cell–cell interactions relevant to treatment outcomes. Results Five major cell clusters were identified: melanoma, keratinocytes, myeloid cells, fibroblasts and inflammatory T/B cells. Complete responders showed strong activation of interferon α/γ pathways, with STAT1, IRF1, IRF9 and NF-κB activated and MYC repressed. VEGFA and HIF signalling suggested a hypoxia-adaptive component. Cell–cell communication analysis revealed major interaction between melanoma-fibroblast in baseline complete responders. Key receptor-ligand associations included ITGB3, SDC2, ERBB3 and ITGB5 in melanoma, CD44 in keratinocytes, LRP1 and ITGA1 in fibroblasts and NTRK1 in immune cells. Post-treatment, MMP7 expression increased in melanoma clusters of complete responders, indicating tissue remodelling. Conclusions These results identify melanoma cell programmes associated with treatment response and fibroblasts mediated pathways as potential therapeutic targets and demonstrate the clinical promise of microwave therapy. Our findings underscore the value of spatial transcriptomics for guiding treatment and support further validation in larger patient cohorts.

COVID-19 continues to present ongoing global health challenges driven by diverse immune responses and heterogeneous clinical outcomes. The ACCORD trial evaluated 3 investigational treatments-bemcentinib, tozorakimab, and zilucoplan-in patients hospitalized with COVID-19, each of which has demonstrated clinical efficacy. To better understand their molecular mechanisms, we conducted a mechanistic follow-up study, integrating transcriptomic and clinical data from 65 patients and applying cellular deconvolution, differential expression, coexpression, and pathway enrichment analyses to uncover treatment-specific immune responses. Each therapy induced transcriptional shifts and modulated distinct immune pathways implicated in severe disease. Bemcentinib primarily modulated myeloid cell populations and inflammatory signalling; zilucoplan enhanced B-cell signalling and lymphocyte-associated pathways; and tozorakimab exerted broad immune and cellular responses across immune cell types. Co-expression analysis revealed gene networks associated with clinical improvement, each driven by distinct treatment-specific hub genes, indicating diverse regulatory mechanisms across treatments. Improved outcomes correlated with gene expression shifts in 4 key immunological pathways: B-cell signalling, antiviral defense, innate inflammation, and platelet/coagulation activity. In contrast, nonresponders had persistent dysregulation of 1 or more of these gene signatures. Our findings define molecular signatures of treatment response and failure in COVID-19, providing mechanistic insight into how distinct therapies modulate the immune system. These insights support the need for adaptive precision medicine approaches tailored to individual, evolving immune trajectories. Moreover, the immunological mechanisms targeted by these repurposed immunomodulatory therapies may inform treatment strategies across a broader spectrum of immune-mediated diseases beyond COVID-19.

Background:Anti-CD20 antibodies are first-line treatments for B cell malignancies. Natural killer (NK) cells are important mediators of anti-CD20 antibody efficacy in humans through antibody-dependent cellular cytotoxicity (ADCC). In B cell malignancies, the lymph nodes are a critical site of pathology and the T cell-derived signals CD40L and IL-4 within the lymph node microenvironment can mediate tumour proliferation, survival and resistance to pro-apoptotic therapy. CD40L and IL-4 have recently been shown to inhibit NK cell activation against chronic lymphocytic leukaemia (CLL) cells via the HLA-E:NKG2A immune checkpoint axis. However, the effect of these signals on NK cell-mediated ADCC of malignant B cells is unclear. Methods:Using a combination of clinical samples, murine models, flow cytometry, immunoblotting, immunohistochemistry, ELISA, bioinformatics and functional assays, we examined the impact of lymph node-mimicking conditions on NK cell-mediated ADCC against malignant B cells. Exogenous CD40L and IL-4 were used to mimic T-B cell interactions in 2D malignant B cell cultures, in addition to a 3D spheroid model of T cell-dependent CLL proliferation. Results:CD40L and IL-4 increased HLA-E expression on the surface of primary CLL cells and non-Hodgkin's lymphoma (NHL) cell lines, and this decreased NK cell-mediated ADCC via ligation of the inhibitory receptor NKG2A. High HLA-E surface expression was observed in lymph node FFPE sections of CLL and NHL patients and in a 3D ex vivo lymph node-mimicking model of CLL. NKG2A blockade potentiated NK cell-mediated ADCC against malignant B cells treated with CD40L and IL-4 and improved anti-CD20 antibody therapy in a murine model of B cell lymphoma. Conclusion:These results reveal a novel mechanism of resistance to anti-CD20 therapy in B cell malignancies and demonstrate that the combination of anti-NKG2A with anti-CD20 could improve the treatment of patients with CLL or NHL.

Despite anti-tuberculous treatment (ATT), central nervous system tuberculosis (CNS-TB) still causes permanent neurological deficits and death. To identify prognostic factors, we profiled a prospective cohort of pediatric HIV-negative tuberculous meningitis (TBM) and non-TBM patients. We found significantly increased cerebrospinal fluid (CSF) matrix metalloproteinases (MMPs) and neutrophil extracellular traps (NETs) in TBM patients with neuroradiological abnormalities and poor outcomes. To dissect mechanisms, we used our existing CNS-TB murine model, which shows neutrophil-rich necrotizing pyogranulomas with MMP-9 and NETs colocalizing, as observed in human CNS-TB pathology. Spatial transcriptomic analysis of both human and murine CNS-TB demonstrates a highly-inflamed and neutrophil-rich microenvironment of inflammatory immune responses, extracellular matrix degradation and angiogenesis within CNS-TB granulomas. Murine CNS-TB treated with ATT and MMP inhibitors SB-3CT or doxycycline show significantly suppressed NETs with improved survival. MMP inhibition arms show attenuated inflammation and well-formed blood vessels within granulomas. Adjunctive doxycycline is highly promising to improve CNS-TB outcomes and survival.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA- sequencing approach in which tissue is reversibly fi xed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate fi ndings using GeoMx whole transcriptome atlas profiling, fi ling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition fi nition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies fi es changes in all major cell types during injury and recovery. We define fi ne the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, fi ltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. fi ltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

BACKGROUND Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODS We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTS TB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSION This biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDING Medical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts.

Langerhans cells (LCs) are distinct among phagocytes, functioning both as embryo-derived, tissue-resident macrophages in skin innervation and repair and as migrating professional antigen-presenting cells, a function classically assigned to dendritic cells (DCs). Here, we demonstrate that both intrinsic and extrinsic factors imprint this dual identity. Using ablation of embryo-derived LCs in the murine adult skin and tracking differentiation of incoming monocyte-derived replacements, we found intrinsic intraepidermal heterogeneity. We observed that ontogenically distinct monocytes give rise to LCs. Within the epidermis, Jagged-dependent activation of Notch signaling, likely within the hair follicle niche, provided an initial site of LC commitment before metabolic adaptation and survival of monocyte-derived LCs. In the human skin, embryo-derived LCs in newborns retained transcriptional evidence of their macrophage origin, but this was superseded by DC-like immune modules after postnatal expansion. Thus, adaptation to adult skin niches replicates conditioning of LC at birth, permitting repair of the embryo-derived LC network.

Matrix stiffening by lysyl oxidase-like 2 (LOXL2)-mediated collagen cross-linking is proposed as a core feedforward mechanism that promotes fibrogenesis. Failure in clinical trials of simtuzumab (the humanized version of AB0023, a monoclonal antibody against human LOXL2) suggested that targeting LOXL2 may not have disease relevance; however, target engagement was not directly evaluated. We compare the spatial transcriptome of active human lung fibrogenesis sites with different human cell culture models to identify a disease-relevant model. Within the selected model, we then evaluate AB0023, identifying that it does not inhibit collagen cross-linking or reduce tissue stiffness, nor does it inhibit LOXL2 catalytic activity. In contrast, it does potently inhibit angiogenesis consistent with an alternative, non-enzymatic mechanism of action. Thus, AB0023 is anti-angiogenic but does not inhibit LOXL2 catalytic activity, collagen cross-linking, or tissue stiffening. These findings have implications for the interpretation of the lack of efficacy of simtuzumab in clinical trials of fibrotic diseases.

Abstract Pancreatic acinar cells are responsible for producing large amounts of digestive enzymes, which are essential for nutrient breakdown but make it difficult to capture the transcriptome of individual cells. To address this challenge, we use a reversible fixative that is compatible with 10X Genomics, thereby preserving the acinar transcriptome and capturing cell composition more faithful to histology. Using this method, we transcriptionally characterize all cell types in the healthy mouse pancreas, and track how these populations change during acute pancreatitis and subsequent recovery. We identify immune cells in the healthy and inflamed pancreas at a level of detail previously unappreciated, characterizing T cell subsets including Th2, γδT, and regulatory T cells. Pancreatitis elicits an influx of neutrophils along with activation and proliferation of macrophages and dendritic cells. Observing significant transcriptional changes in acinar cells after pancreatitis, we define an Acinar to Ductal Metaplasia Index (ADMI), a gene signature that can be used to measure deviation from normal acinar identity in bulk or single cell RNA datasets. We corroborate ADMI using orthogonal GeoMx data, and validate its utility in independent published datasets. We are now expanding our study to perform transcriptomics using our fixation method on pancreatic cancer precursor lesions. Together, we aim to characterize cell types in the mouse pancreas under healthy, inflamed, and precursor conditions to establish a reference atlas for future research on the exocrine pancreas in healthy and diseased states. Citation Format: Katherine J. Aney, Woo-Jeong Jeong, Andres F. Vallejo, Ethan Chen, Austin Wang, Stephanie K. Dougan, Kellie Wise, Kirk Jensen, Luciano Martelotto, Sahar Nissim. A novel approach for exocrine pancreas transcriptomics reveals the cellular landscape of the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A066.

Despite anti-tuberculous treatment (ATT), central nervous system tuberculosis (CNS-TB) still cause permanent neurological deficits and death. To identify prognostic factors, we profiled a prospective cohort of tuberculous meningitis (TBM) and non-TBM patients. We determined significantly increased cerebrospinal fluid (CSF) matrix metalloproteinases (MMPs) and neutrophil extracellular traps (NETs) are up-regulated in TBM patients with neuroradiological abnormalities and poor outcomes. To dissect mechanisms, we created a CNS-TB murine model which show neutrophil-rich necrotizing pyogranulomas with MMP-9 and NETs colocalizing, resembling human CNS-TB. Spatial transcriptomic analysis of both human and murine CNS-TB demonstrates a highly-inflamed and neutrophil-rich microenvironment of inflammatory immune responses, extracellular matrix degradation and angiogenesis within CNS-TB granulomas. Murine CNS-TB treated with ATT and MMP inhibitors SB-3CT or doxycycline show significantly suppressed NETs with improved survival. MMP inhibition arms show attenuated inflammation and well-formed blood vessels within granulomas. Adjunctive doxycycline is highly promising to improve CNS-TB outcomes and survival.

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis.

Background Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored. Objectives Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models. Methods Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models. Results Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins. Conclusions These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema.

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low DNA and RNA quality. In this study, leveraging recent single-cell genomic technology advances, we introduce a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. ### Competing Interest Statement The authors have declared no competing interest. * FFPE : formalin-fixed paraffin-embedded scRNA : seq single-cell RNA-sequencing snRNA : seq single-nucleus RNA-sequencing PBMC : peripheral blood mononuclear cell UMI : unique molecular identifier H&E : hematoxylin and eosin OCT : Optimal Cutting Temperature LSEC : liver sinusoidal endothelial cell NMF : non-negative matrix factorization