γδ T cells are important for host defense at the respiratory mucosa, acting directly or through interactions with other cells. However, how γδ T cells influence other immune cells in the lung remains unclear. Using a genetically engineered mouse model of lung cancer, we show that tumors drive expansion of both CD27+ and CD27- γδ T cells. Advanced microscopy techniques indicated that CD27- γδ T cells are enriched in tumors, whereas CD27+ γδ T cells are more prone to interact with macrophages in tumor-associated adventitial cuffs. SiglecFlow profibrotic airway macrophages were more prevalent in lung tumor-bearing mice than tumor-free mice. This profibrotic subset was reduced in lungs when the cancer model was crossed to Tcrd knockout mice or treated with Vγ1-depleting antibodies but not in TcrgV4/6 knockout mice. Thus, our findings implicate Vγ1 γδ T cells in driving tumor-associated airway macrophage functional imprinting. Determining the translatability to human health may offer new avenues for refining patient management and immunotherapeutic strategies.

Steroid anti-inflammatory drugs, such as dexamethasone, are routinely used to manage brain tumor-associated edema, yet their impact on brain tumor metabolism remains understudied. Here, a metabolomic screen in naïve glioblastoma cells treated with dexamethasone revealed the accumulation of N1-methylnicotinamide, a nicotinamide N-methyltransferase (NNMT) product, through glucocorticoid receptor activation. Using stable isotope-assisted metabolomics in patients with glioblastoma, we showed that nicotinamide conversion into N1-methylnicotinamide exceeds that into NAD+, leading to a ~7-fold accumulation of N1-methylnicotinamide in tumor compared to surrounding brain tissue. In orthotopic models, NNMT activity was enhanced by dexamethasone selectively in glioblastoma tumors but not in contralateral brain. Leveraging the tumor-specific activity of NNMT, we developed a novel 11C-nicotinamide-based positron emission tomography (PET) approach to visualizing glioblastoma tumors. Furthermore, our findings demonstrate that the dexamethasone-induced methionine-dependent nicotinamide methylation becomes detrimental for glioblastoma when combined with a methionine-restricted diet. These results show that steroids rewire methionine and nicotinamide metabolism, enabling the development of innovative PET imaging and metabolic therapies for glioblastoma.

Although 30-40% of human Non-Small Cell lung cancers show low level amplification of c-MYC and genetic evidence supports c-Myc as a key downstream effector of KRas-driven tumourigenesis in mouse models, the functional contribution of MYC to human lung cancer remains unclear. We applied a phenotype-based classifier to the TCGA Lung Adenocarcinoma (LuAd) cohort and found that high MYC transcriptional activity identifies a subset of LuAd with significantly reduced survival. Application of the same methodology to a panel of genetically engineered mouse models identified multiple genotypes that yield the high MYC activity phenotype, disease positioning such models as reflective of distinct subsets of human LuAd. We show that high MYC activity predicts sensitivity to a small molecule dual-inhibitor of the MYC co-factors, EZH2 and G9A, HKMTi-1-005, and that treatment with HKMTi-1-005 strongly reduced MYC protein expression, induced B cell-mediated immune surveillance and suppressed growth of autochthonous KRasG12D-driven lung tumours.

INTRODUCTION:The genes encoding RUNX1 and its binding partner CBFβ are recurrently reported to be mutated in breast cancer, a major cause of mortality in women worldwide. However, the functional role for these proteins remains unproven. METHODS:The putative tumour suppressor role of Runx1 was investigated in genetic mouse models of breast cancer. Stem cell assays, immunohistochemistry and RNAseq analyses were applied to study biological and molecular mechanisms. RESULTS:Runx1 loss of function leads to accelerated disease onset and tumour development in breast cancer models. Combined deletion of Runx1 and Runx2 further resulted in mammary cells becoming exquisitely sensitive to WNT-driven transformation, with expedited emergence of multiple tumours. Runx1 ablation induces a stem cell-like phenotype in mammary epithelial cells, whilst transcriptomic analysis demonstrated activation of multiple oncogenic pathways, especially when Runx2 was co-deleted. Altered Runx expression in the mammary epithelium also drove alterations in the tumour immune microenvironment, with changes to neutrophil and macrophage populations. CONCLUSIONS:Runx1 restricts some forms of breast cancer and inhibits the full oncogenic potential of aberrant WNT signalling. Combined Runx1 and Runx2 loss dramatically accelerates disease progression suggesting that Runx2 can substitute for Runx1 in dampening the oncogenic effects of WNT signalling.

Background Prostate cancer (PC) is the commonest male visceral cancer, and second leading cause of cancer mortality in men in the Western world. Methods Using a forward-mutagenesis Sleeping Beauty (SB) transposon-based screen in a Probasin Cre-Recombinase ( Pb-Cre ) Pten -deficient mouse model of PC, we identified Arid1a loss as a driver in the development of metastatic disease. Results The insertion of transposon in the Arid1a gene resulted in a 60% reduction of Arid1a expression, and reduced tumour free survival ( SB:Pten fl/fl Arid1a INT median 226 days vs SB:Pten fl/fl Arid1a WT 293 days, p = 0.02),with elevated rates of metastasis ( SB:Pten fl/fl Arid1a INT 75% lung metastasis rate vs 17% SB:Pten fl/fl Arid1a WT , p < 0.001). We further generated a Pb-Cre Pten - and Arid1a -deficient mouse model, in which loss of Arid1a demonstrated a profound acceleration in tumorigenesis in Pten fl/fl mice compared to Pten loss alone ( Pb-Cre Pten fl/fl Arid1a +/+ median survival of 267 days vs Pb-Cre Pten fl/fl Arid1a fl/fl 103 days, p < 0.0001). Conclusion Our data revealed homozygous Arid1a loss is required to dramatically accelerate prostate tumourigenesis. Analysis of RNA and ChIP -Sequencing data suggests Arid1a loss enhanced the function of AP-1 subunit cFos. In clinical PC cohort, ARID1A and cFos levels stratified an aggressive subset of PC with a poor survival outcome with a median of only 30 months.

The genes encoding transcription factor RUNX1 and its binding partner CBFB have been reported to be mutated in human breast cancer. Here, we provide evidence that Runx1 loss of function results in accelerated disease onset and tumour development in mouse models of breast cancer, in keeping with a tumour suppressor role for RUNX1 in this disease setting. Combined deletion of Runx1 and the related family member Runx2 resulted in mammary epithelial cells becoming exquisitely sensitive to WNT-driven transformation, with the emergence of multiple tumours early in life. Clonogenic assays indicated that Runx1 ablation induced a stem cell like phenotype in mammary epithelial cells, whilst transcriptome analysis demonstrated activation of multiple oncogenic pathways, especially when Runx2 was co-deleted. Interestingly, altered Runx expression in the mammary epithelium also drove profound alterations in the tumour microenvironment, impacting the immune landscape. These results highlight that Runx1 restricts some forms of breast cancer and inhibits the full oncogenic potential of aberrant WNT signalling. Loss of Runx2 itself did not result in tumour promotion, yet the dramatic effects of combined Runx1 and Runx2 loss suggest that Runx2 can substitute for Runx1 in dampening the oncogenic effects of WNT signalling. ### Competing Interest Statement The authors have declared no competing interest.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer-related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulate progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is broadly independent of the underlying aetiologies4. Despite an increasing range of systemic treatment options for advanced HCC, outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. Here we generated a suite of genetically driven immunocompetent in vivo and matched in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance and metastasis. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with the human histopathology. In a proof-of-principle analysis, we verified response to standard-of-care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not previously been linked to HCC treatment. Cladribine acts in a highly effective subtype-specific manner in combination with standard-of-care therapy.

Cellular senescence is not only associated with ageing but also impacts physiological and pathological processes, such as embryonic development and wound healing. Factors secreted by senescent cells affect their microenvironment and can induce spreading of senescence locally. Acute severe liver disease is associated with hepatocyte senescence and frequently progresses to multi-organ failure. Why the latter occurs is poorly understood. Here we demonstrate senescence development in extrahepatic organs and associated organ dysfunction in response to liver senescence using liver injury models and genetic models of hepatocyte-specific senescence. In patients with severe acute liver failure, we show that the extent of hepatocellular senescence predicts disease outcome, the need for liver transplantation and the occurrence of extrahepatic organ failure. We identify the TGFβ pathway as a critical mediator of systemic spread of senescence and demonstrate that TGFβ inhibition in vivo blocks senescence transmission to other organs, preventing liver senescence induced renal dysfunction. Our results highlight the systemic consequences of organ-specific senescence, which, independent of ageing, contributes to multi-organ dysfunction.

Abstract In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αβ-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27 + Ly6C − cells convert into CD27 + Ly6C + cells, and these CD27 + Ly6C + cells control cancer progression in mice, while the CD27 + Ly6C − cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27 + Ly6C + cells and human Vδ2 + cells, while IL-27 is dispensable for mouse CD27 + Ly6C − cell and human Vδ1 + cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations, with hepatocytes the most likely cell of origin. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies. Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology. In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy.

Background Prostate cancer is the most common cancer in men in the developed world, with most deaths caused by advanced and metastatic disease which has no curative options. Here, we identified Mbtps2 alteration to be associated with metastatic disease in an unbiased in vivo screen and demonstrated its regulation of fatty acid and cholesterol metabolism. Methods The Sleeping Beauty transposon system was used to randomly alter gene expression in the Pten Null murine prostate. MBTPS2 was knocked down by siRNA in LNCaP, DU145 and PC3 cell lines, which were then phenotypically investigated. RNA-Seq was performed on LNCaP cells lacking MBTPS2 , and pathways validated by qPCR. Cholesterol metabolism was investigated by Filipin III staining. Results Mbtps2 was identified in our transposon-mediated in vivo screen to be associated with metastatic prostate cancer. Silencing of MBTPS2 expression in LNCaP, DU145 and PC3 human prostate cancer cells reduced proliferation and colony forming growth in vitro. Knockdown of MBTPS2 expression in LNCaP cells impaired cholesterol synthesis and uptake along with reduced expression of key regulators of fatty acid synthesis, namely FASN and ACACA . Conclusion MBTPS2 is implicated in progressive prostate cancer and may mechanistically involve its effects on fatty acid and cholesterol metabolism.

Background & Aims: Mouse models of lineage tracing have helped to describe the important subpopulations of hepatocytes responsible for liver regeneration. However, conflicting results have been obtained from different models. Herein, we aimed to reconcile these conflicting reports by repeating a key lineage-tracing study from pericentral hepatocytes and characterising this Axin2CreERT2 model in detail.Methods: We performed detailed characterisation of the labelled population in the Axin2CreERT2 model. We lineage traced this cell population, quantifying the labelled population over 1 year and performed in-depth phenotypic comparisons, including transcriptomics, metabolomics and analysis of proteins through immunohistochemistry, of Axin2CreERT2 mice to WT counterparts. Results: We found that after careful definition of a baseline population, there are marked differences in labelling between male and female mice. Upon induced lineage tracing there was no expansion of the labelled hepatocyte population in Axin2CreERT2 mice. We found substantial evidence of disrupted homeostasis in Axin2CreERT2 mice. Offspring are born with sub-Mendelian ratios and adult mice have perturbations of hepatic Wnt/b-catenin signalling and related metabolomic disturbance.Conclusions: We find no evidence of predominant expansion of the pericentral hepatocyte population during liver homeostatic regeneration. Our data highlight the importance of detailed preclinical model characterisation and the pitfalls which may occur when comparing across sexes and backgrounds of mice and the effects of genetic insertion into native loci.(c) 2023 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Dysregulation of the PI3K/AKT pathway is a common occurrence in high‐grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time‐lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P 3 ‐rich and ‐dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase‐activating protein (GAP) AGAP1 and the ECM receptor β1‐integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss‐associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active β1‐integrin to maintain invasive activity into the ECM. The expression of the CYTH2‐ARF6‐AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN‐depleted HGSOC.

6 supplementary Figures with legends under each; 3 Supplementary Tables; Supplementary Methods and References

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

ABSTRACT Immunotherapy is increasingly viewed as treatment of choice for lung cancer, however, clinical responses to immune checkpoint blockade remain highly unpredictable and are largely transient. A deeper mechanistic understanding of the dynamics of tumour:immune interactions is needed to drive rational development of improved treatment strategies. Progress is hampered by a paucity of autochthonous model systems in which to interrogate the 2-way interactions of immune responses to evolving tumours and vice-versa. Specifically, commonly used genetically engineered mouse models typically lack the genetic diversity needed to drive an adaptive immune response. APOBEC mutagenesis signatures are prominent in lung cancer and APOBEC activity is predicted to drive immune visibility through Cytidine deaminase activity, coupled with inaccurate DNA-repair responses. We therefore generated a CRE-inducible APOBEC3B allele, interbred with multiple oncogenic drivers of lung adenocarcinoma, and used the resulting mice to investigate the response to PD1 blockade at single cell resolution. SIGNIFICANCE Using our novel immune-visible model of KRas-driven autochthonous lung adenocarcinoma, we uncovered a surprising increase in tumour-cell expression of EGFR/ERBB ligands following treatment with α-PD1 and present evidence that transient ERBB blockade can restore immune surveillance in KRas mutant LuAd and combine effectively with immune checkpoint blockade.

Protecting mucosal barriers, γδ T cells hold promise for the development of new cancer immunotherapies. In mice, γδ T cells can largely be segregated into CD27 + and CD27 − cells, and their functions are modulated by interactions with surrounding cells. However, which cells communicate directly with γδ T cells in lung adenocarcinoma remains unknown. To address this, we combined flow cytometry, confocal microscopy, and scRNA-seq, using an autochthonous genetically engineered mouse model and different γδ T cell-deficient settings. We found that γδ T cells were increased in tumour-bearing lungs, with an altered phenotype. CD27 − and CD27 + γδ T cells differed in their localisation and interactions including their tropism for macrophages. Overall, we propose a model where CD27 + γδ T cells undermine the differentiation of tumour-associated macrophages into airway macrophages, fostering a negative outcome in lung adenocarcinoma. Determining its translatability to human health may offer new avenues for immunotherapeutic strategies. Summary Guardians of pulmonary homeostasis, γδ T cells remain enigmatic regarding their role in lung adenocarcinoma. Raffo-Iraolagoitia et al. report that a subset of γδ T cells impairs the differentiation of tumour-associated macrophages into airway macrophages, relevant for the outcome of lung adenocarcinoma. Graphical Abstract

Spreadsheet of genes co-regulated by MYC & Miz1 in KMC PDAC cells

Bone marrow mesenchymal stromal cells (MSCs) have immunomodulatory and regenerative potential. However, culture conditions govern their metabolic processes and therapeutic efficacy. Here we show that culturing donor-derived MSCs in Plasmax™, a physiological medium with the concentrations of nutrients found in human plasma, supports their proliferation and stemness, and prevents the nutritional stress induced by the conventional medium DMEM. The quantification of the exchange rates of metabolites between cells and medium, untargeted metabolomics, stable isotope tracing and transcriptomic analysis, performed at physiologically relevant oxygen concentrations (1%O2), reveal that MSCs rely on a high rate of glucose to lactate conversion, coupled with parallel anaplerotic fluxes from glutamine and glutamate to support citrate synthesis and secretion. These distinctive traits of MSCs shape the metabolic microenvironment of the bone marrow niche and can influence nutrient cross-talks under physiological and pathological conditions.