Janin Chandra

Human papillomavirus (HPV)-associated and carcinogen-driven head and neck squamous cell carcinomas (HNSCC) arise through distinct biological routes, yet how each aetiology shapes the tissue state in which carcinogenesis begins remains unclear. We investigated how HPV16 E7 oncoprotein expression and biological sex influence the oral epithelial response before and during carcinogen exposure. Oral epithelium from male and female wild-type (WT) and HPV16 E7-expressing transgenic (K14E7) mice was analysed before and following 4-nitroquinoline-1-oxide (4NQO) induced oral carcinogenesis using flow cytometry, histopathology, immunohistochemistry, and RNA sequencing, with transcriptional programmes compared with human HPV ⁺ and HPV ⁻ HNSCC. Persistent HPV16 E7 expression established epithelial hyperplasia and a baseline transcriptional state enriched for cell-cycle, MYC, interferon response and inflammatory programmes, accompanied by selective remodelling of T cell and myeloid cells. HPV16 E7-driven transcriptional remodelling was more pronounced in females, although pathway enrichment was conserved between sexes. Following 4NQO exposure, WT epithelium underwent transcriptional remodelling that recapitulated programmes already active in untreated K14E7 epithelium, whereas K14E7 epithelium showed limited additional transcriptional change despite similar pathological endpoints. Cross-species analyses showed concordance between the murine WT and K14E7 4NQO model and human HPV ⁻ and HPV ⁺ HNSCC, with sex-associated pathways demonstrating significant, albeit more modest, cross-species concordance. Together, these findings indicate that HPV16 E7 establishes a preconditioned proliferative and inflammatory epithelial state with early immune remodelling that alters responses to carcinogenic injury. HPV16 E7 may redirect rather than accelerate epithelial remodelling during carcinogenesis, with female transcriptional responsiveness to both oncogene expression and carcinogen exposure emerging as a feature of epithelial adaptation.

While immune checkpoint inhibitors (ICIs) have transformed oncology, the determinants of response beyond tumour-intrinsic features remain incompletely understood, particularly the contributions of systemic immunity and the microbiome. Here, in a prospective study of 82 immunotherapy-naïve patients across five solid tumour types, we integrated longitudinal deep immunophenotyping of peripheral blood with gut metagenomic and oral 16S rRNA sequencing. Elevated baseline frequencies of CD5⁺ conventional dendritic cells type 2 (cDC2) and attenuated B cell activation independently predicted clinical benefit, with sustained DC enrichment further associated with durable disease control. Gut metagenomic analysis identified an 11-species responder-associated signature and congruent enrichment of Faecalibacterium MIC7145 at both taxonomic and functional levels. Community-level microbial pathways, including peptidoglycan, UMP, and aromatic compound biosynthesis, correlated with CD5⁺ cDC2 abundance but not with individual species, suggesting that the microbiome’s collective metabolic output may help maintain a priming-competent immune state. Although the oral microbiome was not associated with response, a distinct periodontopathogen signature predicted severe immune-related adverse events. These findings identify CD5⁺ cDC2 as a cornerstone of the antigen-presenting checkpoint and reveal a functional gut microbiome–immune axis that may be exploited to enhance immunotherapy.

Background: Actinic keratoses (AK) arise within chronically UV-damaged skin and are associated with microbial dysbiosis characterised by enrichment of Staphylococcus species and depletion of the commensal bacterium Cutibacterium acnes. Whether targeted reintroduction of a beneficial commensal can durably reprogramme the dysbiotic cutaneous microbiome remains unknown. We evaluated the capacity of topical C. acnes strain D1, a live commensal biotherapeutic, to modify microbial communities within AK lesions and the surrounding photodamaged skin field.Methods: We conducted a randomised, double-blind, placebo-controlled pilot trial in adults with clinically diagnosed AK. Participants were randomised (1:1) to receive either an anhydrous topical formulation containing live C. acnes D1 or placebo, applied twice daily for 14 days to a target AK lesion. Microbiome samples were collected from treated lesions and contralateral untreated photodamaged skin at baseline and up to 180 days following treatment. Small-subunit ribosomal RNA (SSU rRNA) sequencing was used to assess microbial composition, diversity, and taxonomic abundance. Analyses followed intention-to-treat principles.Results: Twenty participants were randomised (10 C. acnes D1, 10 placebo). Topical administration of C. acnes D1 induced sustained microbiome remodelling that persisted for 120–180 days after treatment cessation. Compared with placebo, treatment reduced the relative abundance of Staphylococcus species, increased microbial diversity, and promoted broader compositional restructuring towards a commensal-enriched microbial community. Similar ecological shifts were observed in contralateral untreated photodamaged skin, indicating field-wide microbiome effects following localised intervention. No treatment-related adverse events were observed.Conclusions: This study provides the first controlled clinical evidence that a short-course topical live commensal biotherapeutic can induce durable and field-wide microbiome reprogramming in human skin. These findings demonstrate the potential of commensal-based microbial ecological engineering to sustainably modify disease-associated microbiome states and support further investigation of microbiome-directed interventions for actinic keratosis and other chronic cutaneous disorders.

IL-33, an alarmin cytokine binding to IL1RL1 (ST2) receptor, plays a controversial role in cancer. Using an HPV16-E7 oncoprotein-mediated murine model of squamous intraepithelial hyperplasia, a pre-stage of squamous cell carcinoma (SCC), we characterised the interactions of hyperproliferative epithelial cells and immune cells regulated by IL-33 and IL1RL1. We show that basal epithelial cells in epithelial hyperplasia co-express IL-33 and genes involved in MHC class II antigen presentation and in epithelial stress response. Using spatial transcriptomics, we demonstrate that IL1RL1+Foxp3+Tregs are in close proximity to IL-33-expressing cells, in both the murine model and in human cervical intraepithelial neoplastic tissues. Cytoplasmic location of IL-33 is associated with its bioactivity, and we show that a subset of epithelial cells in epithelial hyperplasia contains high levels of cytoplasmic IL-33. Genetic deletion of the IL1RL1 gene led to partial rejection of HPV16-E7-expressing skin grafts, demonstrating that IL1RL1 plays a functional role in mediating immune suppression in epithelial hyperplasia. IL1RL1-deletion further led to a significant reduction in Tregs in epithelial hyperplasia. The IL1RL1 signalling pathway, therefore, represents a potential therapeutic target for intraepithelial hyperplasia and SCC.

Human cutaneous squamous cell carcinomas (SCCs) and actinic keratoses (AK) display microbial dysbiosis with an enrichment of staphylococcal species, which have been implicated in AK and SCC progression. SCCs are common in both felines and canines and are often diagnosed at late stages leading to high disease morbidity and mortality rates. Although recent studies support the involvement of the skin microbiome in AK and SCC progression in humans, there is no knowledge of this in companion animals. Here, we provide microbiome data for SCC in cats and dogs using culture-independent molecular profiling and show a significant decrease in microbial alpha diversity on SCC lesions compared to normal skin ( P ≤ 0.05). Similar to human skin cancer, SCC samples had an elevated abundance of staphylococci relative to normal skin—50% (6/12) had >50% staphylococci, as did 16% (4/25) of perilesional samples. Analysis of Staphylococcus at the species level revealed an enrichment of the pathogenic species Staphylococcus felis in cat SCC samples, a higher prevalence of Staphylococcus pseudintermedius in dogs, and a higher abundance of Staphylococcus aureus compared to normal skin in both companion animals. Additionally, a comparison of previously published human SCC and perilesional samples against the present pet samples revealed that Staphylococcus was the most prevalent genera across human and companion animals for both sample types. Similarities between the microbial profile of human and cat/dog SCC lesions should facilitate future skin cancer research. IMPORTANCE: The progression of precancerous actinic keratosis lesions (AK) to cutaneous squamous cell carcinoma (SCC) is poorly understood in humans and companion animals, despite causing a significant burden of disease. Recent studies have revealed that the microbiota may play a significant role in disease progression. Staphylococcus aureus has been found in high abundance on AK and SCC lesions, where it secretes DNA-damaging toxins, which could potentiate tumorigenesis. Currently, a suitable animal model to investigate this relationship is lacking. Thus, we examined the microbiome of cutaneous SCC in pets, revealing similarities to humans, with increased staphylococci and reduced commensals on SCC lesions and peri-lesional skin compared to normal skin. Two genera that were in abundance in SCC samples have also been found in human oral SCC lesions. These findings suggest the potential suitability of pets as a model for studying microbiome-related skin cancer progression.

Objectives: Langerhans cells (LCs) are epithelial antigen‐presenting cells (APC) contributing to immune surveillance. LCs depend on interleukin 34 (IL34) production by epithelial cells. This study aimed to uncover mechanisms of alteration of IL34 and LC function in squamous cell carcinoma (SCC).Methods: Cancer cohort data were used to identify associations between SCC and IL34. ATAC‐seq of keratinocytes (KCs) and LCs from a murine model of epithelial hyperplasia, driven by HPV16 E7 oncoprotein (K14E7), was analysed. Transcriptomic data were used to validate findings. RNAscope, RT‐qPCR, ELISA and confocal imaging was used to analyse IL34 expression and LCs in a spatial context.Results: IL34 mRNA is downregulated in human SCCs of the head and neck, the cervix, the lung and the oesophagus, and low IL34 expression is associated with poor survival. We demonstrate that KCs of K14E7 mice have reduced Il34 gene accessibility, mRNA and protein, as well as broad changes in promotor accessibility associated with cell adhesion and immune responses. Chromatin accessibility was substantially changed in LCs, including increased accessibility of the Csf1r gene, and changes in promotors associated with cytoskeleton arrangement and antigen processing and presentation. We discovered altered spatial LC dendrite organisation in hyperproliferative epithelium.Conclusion: Squamous cell carcinoma of the cervix, head and neck, oesophagus and lung demonstrate downregulation of IL34, which is associated with poor survival, and with alterations in LC spatial organisation and function. These findings suggest that reduced IL34 expression in SCC may contribute to impaired local immunity through LC dysregulation.

Cutaneous squamous cell carcinoma (cSCC) and its premalignant precursor, actinic keratosis (AK), present a global health burden that is continuously increasing despite extensive efforts to promote sun safety. Chronic UV exposure is a recognized risk factor for the development of AK and cSCC. However, increasing evidence suggests that AK and cSCC is also associated with skin microbiome dysbiosis and, in particular, an overabundance of the bacterium Staphylococcus aureus (S. aureus). Studies have shown that S. aureus-derived toxins can contribute to DNA damage and lead to chronic upregulation of proinflammatory cytokines that may affect carcinogenesis. Eradication of S. aureus from AK lesions and restoration of a healthy microbiome may therefore represent a therapeutic opportunity to alter disease progression. Whilst antibiotics can reduce the S. aureus load, antibiotic resistant S. aureus pose an increasing global public health threat. The use of specific topically delivered probiotics has been used experimentally in other skin conditions to restore eubiosis, and could therefore also present a non-invasive treatment approach to decrease S. aureus colonization and restore a healthy skin microbiome on AK lesions. This article reviews mechanisms by which S. aureus may contribute to cutaneous carcinogenesis, and discusses hypotheses and theories that explore the therapeutic potential of specific bacterial species which compete with S. aureus in an attempt to restore microbial eubiosis in skin.

Integration of high-dimensional tumor gene expression data with clinicopathological data can increase our understanding of disease diversity, enable retrospective patient stratification, and identify new potential biomarkers and therapeutic targets. Using a systems biology approach, we provide a holistic overview of gene co-expression networks in head and neck squamous cell carcinomas (HNSCC). Weighted gene co-expression network analysis of HNSCC RNA sequencing data from 519 patients from The Cancer Genome Atlas (TCGA) was used to determine correlates of 5-year survival, using regression tree-based optimal threshold calculations. Survival-associated gene sets were transformed to gene set scores that were assessed for correlation with clinicopathological data. We identified 8 gene co-expression modules for HNSCC tumors, each of which contained co-expressed genes associated significantly with 5-year survival. Survival-associated co-expression gene signatures correlated dominantly with tumor HPV and p16 status. Network analysis identified that survival was associated with signaling networks of infection, immunity, epithelial-mesenchymal transition (EMT), hypoxia, glycolysis, focal adhesion, extracellular matrix, MYC signaling, autophagy and transcriptional regulation. EMT-associated gene signatures were expressed dominantly in fibroblasts, and cancer-associated fibroblasts were inversely correlated with immune activity. Interestingly, a high Immune Suppression Score based on expression of 21 genes associated with immune inhibition and including immune checkpoints, cytokines and regulatory T cell factors, was also associated with increased survival probability, and was significantly higher in HPV+ HNSCC. Networks associated with HNSCC survival were further associated with survival in cervical cancer, melanoma and lung cancer. This study defines 5129 genes associated with HNSCC survival, organized into co-expressed networks, their correlation with clinicopathological data, and with gene expression data from other malignant diseases, and provides a source for the discovery of biomarkers and novel therapies for HNSCC.