Spatial transcriptomics enables the understanding of the spatial architecture of tissues, providing deeper insight into tissue structure and cellular neighbourhoods. A crucial step in the analysis of spatial data is cell type identification. In single cell RNA-sequencing (scRNA-seq) analysis, cells are clustered according to their transcriptional similarity, and marker genes for each cluster identified. Marker analysis identifies genes highly expressed in each cluster compared to the remaining clusters, and these marker genes are used to annotate clusters with cell types. For spatial data, there are limited software tools for appropriate marker gene detection methods that account for the spatial distribution of gene expression. Tools developed for scRNA-seq ignore spatial information for the cells and genes. We have developed a hybrid approach to prioritize marker genes that uses the spatial coordinates of gene detections and cells making up clusters. We propose a binning approach that effectively “pseudobulks” gene detections and cells within clusters that can then be used as input into linear models for marker analysis. Our approach can account for multiple samples and background noise. We have tested our methods on several public datasets from different platforms including Xenium, CosMx and MERSCOPE. The marker genes detected by our method show strong spatial correlation with the corresponding clusters and have increased specificity compared to other methods. The method is implemented in the jazzPanda R Bioconductor package and is publicly available ( https://bioconductor.org/packages/jazzPanda ).

Tumour-infiltrating FOXP3+ regulatory T cells (Tregs) exert suppression of anti-tumour immunity in non-small cell lung cancer (NSCLC), contributing to poor prognosis and immunotherapy resistance. The BCL-2 family pro-survival protein, MCL-1, is a critical controller of lymphoid Treg viability, yet its role in tumour-infiltrating Tregs remains poorly defined. Here we find that tumour-infiltrating effector Tregs in human NSCLC exhibit an activation-associated shift in BCL-2 family pro-survival protein expression typified by elevated MCL-1 expression. Pharmacological inhibition of MCL-1 with the BH3 mimetic S63845 induced moderate apoptotic cell death in both human and murine tumour-infiltrating Tregs, coincident with transient enhancement of CD8⁺ T cell activity. Combined MCL-1 inhibition and anti-PD1 immunotherapy further reduced tumour-infiltrating effector Treg abundance and influenced CD8⁺ T cell dynamics, although these effects were not sufficient to extend long-term survival. Mechanistically, we found that IL-33 upregulated MCL-1 expression and was required to support activated tumour-infiltrating Tregs. These results establish MCL-1 as an important regulator for tumour-infiltrating Treg survival and highlight the potential of repurposing BH3 mimetics to modulate immune suppression in NSCLC.

The tumor microenvironment is composed of diverse immune populations that can either support anti-tumor immunity or promote tumor progression. Myeloid cells are major drivers of immunosuppression, yet therapies targeting them have shown limited success. To uncover mechanisms underlying myeloid-driven immune suppression, we performed spatial multi-omics analyses of non-small cell lung cancer (NSCLC). Independent of oncogenic driver status, tumors stratify into lymphoid-enriched, myeloid-enriched, and mixed immune-infiltrated subtypes. In tumor and adjacent non-malignant lungs, we identify myeloid-instructed CD14+CD4+ T cells. These cells arise through trogocytosis adopting an atypical phenotype. In lymphoid-enriched tumors, high infiltration of CD14+CD4+ T cells correlates with poor patient survival. Spatial transcriptomics reveal enrichment of tumor necrosis factor alpha (TNF-α) signaling in CD14+CD4+-T-cell-rich tumors. Functional assays demonstrate that TNF-⍺ enhanced trogocytosis, promoting the formation of CD14+CD4+ T cells. These findings uncover a TNF-⍺-mediated mechanism of immunosuppression in the TME and highlight aberrant myeloid-T cell interactions as contributors to NSCLC progression.

The COVID-19 pandemic has highlighted the long-term impact of viral infections on health, yet the impact of SARS-CoV-2 infection on lung cancer development has remained unclear. In a recent study published in Cell, Qian and colleagues describe an increased risk for the development of lung cancer in patients who suffered from past severe COVID-19. To dissect the mechanisms underlying the epidemiologic evidence, the authors used multiple murine cancer models to demonstrate that respiratory infections, including flu and SARS-CoV-2 infections, remodel the lung tissue microenvironment, leading to an accumulation of neutrophils and immunosuppressive cytokines that promote and sustain a tumor-prone environment. Tumors from previously infected mice contained not only increased numbers of protumorigenic neutrophils but also CD8+ T cells that showed more pronounced signs of immune exhaustion. SARS-CoV-2 vaccination reduced the increase in cancer growth, and blocking neutrophil infiltration combined with immune checkpoint blockade reversed the increase in lung tumor burden. This study demonstrates a long-term molecular memory, or epigenetic imprinting, induced by viral infections in both epithelial and immune cells that can accelerate oncogenesis, and highlights how vaccination protects not only against acute viral infections but also limits the long-term impact of infection on future cancer development.

Supplementary Table 4: Systemic therapy received by each patient prior to recruitment into the trial.

Supplementary Figure 5. Functional markers expressed in CD8+ T cells in gPFS patients

Supplementary Table 5. CAVATAK samples: time between biopsies and biopsy sites.

Supplementary Figure 2. Tumor cell characteristics at baseline and on treatment in gPFS and loPFS patients

PURPOSE:Acquired or de novo resistance to immune checkpoint inhibitors occurs in the majority of advanced non-small cell lung cancers. There is an unmet need to improve outcomes for patients with this condition. Oncolytic viruses represent an attractive treatment approach because of their dual activity in inducing tumor cell lysis directly and potentially augmenting antitumor immunity. In this study, we present the safety, efficacy, and translational findings from a phase I/II single-arm trial utilizing CVA21, an oncolytic coxsackievirus, in combination with pembrolizumab in patients with advanced pretreated non-small cell lung cancers. PATIENTS AND METHODS:We performed paired pre- and posttreatment biopsies in 10 patients who received intravenous CVA21 and pembrolizumab, eight of whom had prior treatment with immune checkpoint inhibitor therapy. Whole-genome sequencing and spatial proteomics were performed to comprehensively characterize the response to CVA21. RESULTS:Combination CVA21/pembrolizumab (anti-PD-1) therapy was well tolerated with no serious treatment-related adverse events. Partial responses were seen in two patients with prior acquired anti-PD-1 resistance and disease stabilization in six patients, giving a clinical benefit rate of 80%. High baseline tumor mutational burden and PD-L1 expression were observed in patients with better response to treatment. Interestingly, an increase in antigen presentation and CD8+ T-cell infiltration was observed on-treatment compared with baseline in patients with better progression-free survival. CONCLUSIONS:This study demonstrates the potential of CVA21 to modulate the immunogenicity of tumor cells and remodel the tumor microenvironment, providing insights for patient selection for trials involving novel immunotherapeutic approaches.

Long-read RNA sequencing enables full-length transcript profiling and improved isoform resolution, but variable platforms and evolving chemistries demand careful benchmarking for reliable application. We present LongBench , a matched, multi-platform reference dataset spanning bulk, single-cell, and single-nucleus transcriptomics across eight human lung cancer cell lines with synthetic spike-in controls. LongBench incorporates three state-of-the-art long-read protocols alongside Illumina short reads: Oxford Nanopore Technologies (ONT) PCR-cDNA, ONT direct RNA, and PacBio Kinnex. We systematically evaluate transcript capture, quantification accuracy, differential expression, isoform usage, variant detection, and allele-specific analyses. Our results show high concordance in gene-level differential analyses across protocols, but reduced consistency for transcript-level and isoform analyses due to lengthand platform-dependent biases. Single-cell long-read data are highly concordant with bulk for high-confidence features, though single-nuclei data show reduced feature detection. LongBench provides one of the largest publicly available long-read benchmarking resources, enabling rigorous cross-platform evaluation and guiding technology selection for transcriptomic research. ### Competing Interest Statement The authors acknowledge the support of both PacBio and Oxford Nanopore Technologies who provided sequencing reagents used in the single-cell / single-nuclei arm of this study. Y.Y, K.Z, M.B.C. and Q.G. received travel support from Oxford Nanopore Technologies to attend conferences. Q.G. received travel support from PacBio to attend a conference. The authors have no other competing interests, and the Funders had no involvement in study design, data analysis, interpretation and writing of the article, or the decision to submit the work for publication. Australian National Health and Medical Research Council Medical Research Future Fund Researcher Exchange and Development in Industry Fellowship Research Foundation-Flanders Dutch MS Research Foundation KU Leuven (BOF-FKO, Bijzonder Onderzoeksfonds – Fundamenteel Klinisch Onderzoeker) Victorian State Government Operational Infrastructure Support Australian Cancer Research Foundation

Supplementary Figure 1. Survival analysis.

Supplementary Figure 3. Analysis of immune cell populations in all samples

Supplementary Table 2. Representativeness of study participants

Supplementary Figure 4. Analysis of immune cell populations in gPFS and loPFS samples.

Supplementary Table 3: Antibodies used in the MIBI panel.

The mechanisms governing the progression of precursor lesions to invasive lung adenocarcinoma (LUAD) remain poorly understood. In this issue of Cancer Cell, Zhu et al. map the dynamic immune changes associated with this progression using high-resolution spatial mapping. Their identification of TIM-3 as a potential target may shift strategies for LUAD immunoprevention.

In vivo models that replicate and reproduce human lung cancer and its response to therapy are necessary for the development of new therapeutic strategies and understanding drug resistance. Imaging lung tumors in live animals to monitor tumor growth and response to therapy is challenging owing to the location of the lungs and their constant movement during breathing. X-ray velocimetry (XV) is a novel functional lung imaging technique that maps regional lung expansion during breathing, providing spatial information on where ventilation changes occur. The aim of this pilot study was to use XV and flexiVent lung mechanics assessments to determine the effect of tumor growth on lung function in mice at 2 or 3 weeks post tumor induction and to evaluate the efficacy of these two tools. Histological analysis showed that tumor growth was not uniform between animals. At 3 weeks post tumor induction, some XV ventilation and flexiVent lung mechanics parameters were significantly different from baseline. Both techniques gave metrics that correlated with the tumor counts from the histology. In some mice, XV revealed localized regions with altered expansion rates.

Inflammation and excess cytokine release are hallmarks of severe COVID-19. While programmed cell death is known to drive inflammation, its role in SARS-CoV-2 pathogenesis remains unclear. Using gene-targeted murine COVID-19 models, we here find that caspase-8 is critical for cytokine release and inflammation. Loss of caspase-8 reduces disease severity and viral load in mice, and this occurs independently of its apoptotic function. Instead, reduction in SARS-CoV-2 pathology is linked to decreased IL-1β levels and inflammation. Loss of pyroptosis and necroptosis mediators in gene-targeted animals provides no additional benefits in mitigating disease outcomes beyond that conferred by loss of caspase-8. Spatial transcriptomic and proteomic analyses of caspase-8-deficient mice confirm that improved outcomes are due to reduced pro-inflammatory responses, rather than changes in cell death signalling. Elevated expression of caspase-8 and cFLIP in infected lungs, alongside caspase-8-mediated cleavage of N4BP1, a suppressor of NF-kB signalling, indicates a role of this signalling axis in pathological inflammation. Collectively, these findings highlight non-apoptotic functions of caspase-8 as a driver of severe COVID-19 through modulation of inflammation, not through the induction of apoptosis.