The acidic tumor microenvironment (TME) favors cancer aggressiveness via incompletely understood pathways. Here, we asked whether adaptation to environmental acidosis (pH 6.5) selects for human pancreatic cancer stem cell (CSC) properties. RNA sequencing (RNA-seq) of acid-adapted (AA) Panc-1 cells revealed CSC pathway enrichment and upregulation of CSC markers. AA Panc-1 cells exhibited classical CSC characteristics including increased aldehyde dehydrogenase (ALDH) activity and b-catenin activity. Panc-1, PaTu8988s, and MiaPaCa-2 cells all exhibited increased pancreatosphere-forming efficiency after acid adaptation but differed in CSC marker expression and did not exhibit typical flow cytometric CSC populations. However, single-nucleus sequencing revealed the acid adaptation-induced emergence of Panc-1 cell subpopulations with clear CSC characteristics. In orthotopic mouse tumors, AA Panc-1 cells exhibited enhanced aggressiveness, liver and lung metastasis, compared to controls. Collectively, our work suggests that acid adaptation enriches for pancreatic CSC phenotypes with unusual traits via several trajectories, providing new insight into how acidic microenvironments favor cancer aggressiveness.

Cellular plasticity and transitional cellular states are crucial for tissue regeneration across multiple organs. In the pancreas, oncogenic Kras hijacks this program, acting on tissue-specific enhancers to prevent the resolution of acinar-to-ductal metaplasia (ADM) and lock regeneration into a pro-inflammatory state that progresses to cancer. Enhancer transcription, an early event during cellular state transitions, can generate stable enhancer-associated long noncoding RNAs (lncRNAs) positioned near key transcription factors and chromatin contact boundaries, often enriched for disease-associated variants. While enhancer-associated lncRNAs have been implicated in transcriptional regulation and genome organization, their role in pancreas regeneration and cancer initiation has remained unexplored. In this study, we investigated the expression of epithelial long noncoding RNAs (lncRNAs) and their target genes in PDAC precursor lesion formation. We focus on lncRNAs transcribed from enhancer elements near cell identity transcription factors. We demonstrate that LINC00673, expressed from a Sox9-associated super-enhancer during pancreatic development, is reactivated in PDAC. Conditional deletion of LINC00673 in the murine pancreatic epithelium accelerates resolution of ADM and significantly impairs PDAC initiation. Notably, LINC00673 harbors a variant associated with risk of developing PDAC. Our study identifies a critical function of LINC00673 in regulating both cell-autonomous and non-cell-autonomous processes during pancreas regeneration and Kras-driven cancer initiation. Furthermore, we highlight a previously unrecognized role of transcribed super-enhancers in facilitating long-range gene regulation during pancreatic cancer initiation. These findings reveal a novel regulatory layer linking developmental enhancer activity, cellular plasticity and pancreatic disease progression. Teaser Long noncoding RNAs from developmental enhancers play a role in long-range gene regulation with crucial biological impacts in development and cancer. Grant support This project has been supported by the Danish Cancer Society (R302-A17481). LA is supported by core funding of the Biotech Research and Innovation Center, the Danish Cancer Society (R302-A17481, R322-A17.350), The Novo Nordisk Foundation (NNF21OC0070884) and The Innovation Fund (Eurostars 2807). The Novo Nordisk Foundation Center for Stem Cell Biology was supported by Novo Nordisk Foundation grants NNF17CC0027852. ### Competing Interest Statement The authors have declared no competing interest. * ADM : Acinar-to-ductal metaplasia PDAC : pancreatic ductal adenocarcinoma PanIN : pancreatic intraepithelial neoplasia IPMN : intraductal papillary mucinous neoplasm lncRNAs : long noncoding RNAs Kras : Kirsten rat sarcoma proto-oncogene ISH : in situ hybridization hPSC : human pluripotent stem cells CAE : caerulein Hi-C : high-throughput chromosome conformation capture scRNAseq : single cell RNAseq

The tumor microenvironment (TME) plays critical roles in cancer development, aggressiveness, and treatment resistance. The TME comprises cellular (stromal) components as well as gradients of physicochemical properties, including hypoxia and acidosis. Understanding of how hypoxia and acidosis gradients impact cancer phenotypes is lacking, in large part due to challenges in precisely mimicking and controlling such gradients in a manner compatible with the growth of cancer- and stromal cells. Here, we design and validate a microfluidic device enabling orthogonal gradients of oxygen and pH. Both gradients are established by diffusion from a nearby source and sink in the observation area in the absence of flow. This produces linear gradients at steady state. Our device enables a wide range of spatiotemporally resolved analyses, from ‘omics to live cell imaging, interrogating the impact of the physicochemical TME on disease development. The design is easily adaptable, making it valuable for a wide range of questions involving physicochemical gradients. ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, https://ror.org/04txyc737, NNF19OC0057739, NNF21OC0069598 Carlsberg Foundation, https://ror.org/01kpjmx04, CF20-0491

STUDY QUESTION:Does the transcriptome of preconceptional endometrium in the proliferative phase show a specific profile in women with recurrent pregnancy loss (RPL)? SUMMARY ANSWER:A specific differential gene expression signature in endometrial samples for women experiencing RPL compared with IVF control women was identified including an RPL subgroup characterized by upregulation of immune-related genes and pathways. WHAT IS KNOWN ALREADY:RPL affects 1-3% of couples trying to become parents with both short- and long-term health implications; furthermore, the underlying pathophysiology is complex and heterogeneous with no explanations found for more than half of the couples. Some studies indicate that immunological dysfunction plays a role even preconceptionally and during implantation; however, the few published studies of endometrial transcriptomes from women with RPL have had small sample sizes and focused on the secretory phase of the menstrual cycle. STUDY DESIGN, SIZE, DURATION:The study was based on two cohorts of women: an RPL cohort (n = 108) and a control cohort (n = 27). Endometrial samples were collected at two university hospital clinics from March 2013 until February 2019. Dating of the endometrium was made by histological examination by experienced pathologists. PARTICIPANTS/MATERIALS, SETTING, METHODS:All women were between 18 and 42 years at the time of collection of the biopsy. RPL was defined as three or more consecutive pregnancy losses or two second-trimester losses or stillbirths. The control group consisted of women referred to IVF/ICSI treatment with a presumed healthy endometrium. All biopsies, except one, were collected in a natural menstrual cycle. In total, 108 women with RPL were subjected to RNA-seq analysis. Seventy-six biopsies were in the proliferative phase, 29 were in the secretory phase, and three could not be classified. For the control women, in total, 27 were included in the RNA-seq analysis; 22 biopsies were in the proliferative phase, one was in the secretory phase, and four could not be classified. Total RNA was extracted from the endometrial biopsies, which had been stored in RNA stabilization solution at -80°C. RNA-seq reads were mapped and quantified using a reference transcriptome and analysed using principal component analysis (PCA), hierarchical clustering, and differential gene expression methods. MAIN RESULTS AND THE ROLE OF CHANCE:PCA showed a clear separation of biopsies collected either in the proliferative or secretory phase. For the main analyses, we focused on the women with biopsies in the proliferative phase. PCA and differentially expressed genes (DEGs) revealed that RPL patients were characterized by upregulation of a limited number of immune-related genes and pathways. Further analyses revealed that subjects could describe a gene expression continuum, separable into four different subgroups by the gene expression data, where one subgroup consisted only of IVF controls, one was mixed, and two were composed of RPL patients only. The final analyses showed a distinct subgroup in the RPL cohort with stronger upregulation of immune-related genes, and deconvolution analyses of the bulk RNA-sequencing data together with immunohistochemistry analyses of the CD56 marker indicated an increased number of natural killer cells in this subgroup. A machine-learning model based on the Random Forest algorithm and gene expression data from the 157 DEGs (RPL vs controls) was trained on a subset of the cohort and validated using the remaining subjects, reaching on average 96.6% accuracy (95% CI: 93.3-96.7%), 95.7% sensitivity (95% CI: 95.7-95.7%), and 99.5% specificity (95% CI: 85.7-100.0%). The same analysis using only the most informative genes increased validation accuracies further. LIMITATIONS, REASONS FOR CAUTION:The size of the IVF control group and difficulties in defining the most optimal type of control group is a recurrent limitation in this and other RPL studies. Some of the women from the control group might be subfertile in relation to endometrial factors. However, the current control group is a mix of women with different pregnancy and fertility records, which is a strength. Alternative control groups could be women with one to two healthy pregnancies or one to two pregnancy terminations. Furthermore, in only about 10% of the RPL cases information on foetal pathology or chromosomal aneuploidy was obtained. WIDER IMPLICATIONS OF THE FINDINGS:The findings presented here indicate that a gene expression signature exists, which can be used to classify RPL patients versus control (non-RPL) women. An interesting aspect is that a pregnancy loss in some women thereby might result in a specific signature detectable as a specific endometrial gene expression profile possibly irrespectively of the cause of the pregnancy loss. Aside from contributing to a further understanding of the pathophysiology and development of new treatments, this finding could lead to a specific and cost-effective test that at an early stage could identify women with high risk of experiencing RPL. To this end, further perspectives include a prospective study, which would explore further utility of the predictive model by analysing endometrial samples collected in the proliferative phase from a cohort of women, who have experienced one pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the Region Zealand Health Sciences Research Foundation, Zealand University Hospital through the ReproHealth Research Consortium ZUH, the Frimodt-Heineke Foundation, 'Direktør Emil C. Hertz og Hustru Inger Hertz' Fond', the Torben and Alice Frimodt's Foundation, the Novo Nordisk Foundation, and the Independent Research Fund Denmark. L.H.J.A., R.S.M., Y.D., K.J.H., H.S.N., A.S., and T.V.F.H. are inventors on a patent application (number EP24171923.6) submitted by Region Zealand, The Capital Region of Denmark, and the University of Copenhagen that covers a diagnostic test based on the results of the study. The authors declared no other competing interests. TRIAL REGISTRATION NUMBER:N/A.

Ulcerative colitis (UC) is characterized by chronic relapsing inflammation starting from the rectum and distal colon, which in severe disease cases may affect the entire colon. Intestinal stem cells (ISCs) directly isolated from inflamed UC colonic tissue specimens have been found to present an inflammatory gene expression profile. However, a critical issue is whether these cells retain memory of exposure to inflammation and/or therapeutics. Here, we aimed to investigate whether human intestinal epithelial cells retain the inflammatory state observed in vivo when expanded in vitro as 3D cultured organoids to assess their suitability for therapeutic transplantation. ISCs were isolated from noninflammatory bowel disease controls (noninflamed; n = 18), as well as from colonoscopy-obtained biopsies of the sigmoid colon from individuals diagnosed with UC (inflamed), who were glucocorticoid naïve (n = 19). Moreover, ISCs were collected from all patients with inflammatory bowel disease following prednisolone treatment. Epithelial cells were cultured as 3D intestinal organoids in media to support stem cell maintenance and differentiation. Subsequently, the 3D intestinal organoids were harvested at the end of passage 2 for bulk RNA sequencing. The data revealed that the cellular phenotype of in vitro-cultured epithelial cells isolated from inflamed tissue did not maintain the hallmarks of inflammation observed in the ulcerated environment from which the cells were initially obtained. Our findings indicate that the autologous reinsertion of in vitro-expanded ISCs in active stages of UC may aid in intestinal healing, which calls for future clinical studies. Additionally, a link between organoid morphology and the inflammatory state of the tissue of origin was identified, as organoids derived from inflamed colon exhibited a lower degree of circularity.

Analysis of transcript function is greatly aided by knowledge of the full-length RNA sequence. New long-read sequencing enabled by Oxford Nanopore and PacBio devices have the potential to provide full-length transcript information; however, standard methods still lack the ability to capture true RNA 5' ends and select for polyadenylated (pA+) transcripts only. Here, we present a method that, by utilizing cap trapping and 3'-end adapter ligation, sequences transcripts between their exact 5' and 3' ends regardless of polyadenylation status and without the need for ribosomal RNA depletion, with the ability to characterize polyadenylation length of RNAs, if any. The method shows high reproducibility, can faithfully detect 5' ends, 3' ends and splice junctions, and produces gene-expression estimates that are highly correlated to those of short-read sequencing techniques. We also demonstrate that the method can detect and sequence full-length nonadenylated (pA-) RNAs, including long noncoding RNAs, promoter upstream transcripts, and enhancer RNAs, and present cases where pA+ and pA- RNAs show preferences for different but closely located transcription start sites. Our method is therefore useful for the characterization of diverse capped RNA species and analysis of relationships between transcription initiation, termination, and RNA processing.

Genome annotation files play a critical role in dictating the quality of downstream analyses by providing essential predictions for gene positions and structures. These files are pivotal in decoding the complex information encoded within DNA sequences. Here, we generated experimental data resolving RNA 5'- and 3'-ends as well as full-length RNAs for cassava TME12 sticklings in ambient temperature and cold. We used these data to generate genome annotation files using the TranscriptomeReconstructoR (TR) tool. A careful comparison to high-quality genome annotations suggests that our new TR genome annotations identified additional genes, resolved the transcript boundaries more accurately and identified additional RNA isoforms. We enhanced existing cassava genome annotation files with the information from TR that maintained the different transcript models as RNA isoforms. The resultant merged annotation was subsequently utilized for comprehensive analysis. To examine the effects of genome annotation files on gene expression studies, we compared the detection of differentially expressed genes during cold using the same RNA-seq data but alternative genome annotation files. We found that our merged genome annotation that included cold-specific TR gene models identified about twice as many cold-induced genes. These data indicate that environmentally induced genes may be missing in off-the-shelf genome annotation files. In conclusion, TR offers the opportunity to enhance crop genome annotations with implications for the discovery of differentially expressed candidate genes during plant-environment interactions.

The fetal development of organs and functions is vulnerable to perturbation by maternal inflammation which may increase susceptibility to disorders after birth. Because it is not well understood how the placenta and fetus respond to acute lung- inflammation, we characterize the response to maternal pulmonary lipopolysaccharide exposure across 24 h in maternal and fetal organs using multi-omics, imaging and integrative analyses. Unlike maternal organs, which mount strong inflammatory immune responses, the placenta upregulates immuno-modulatory genes, in particular the IL-6 signaling suppressor Socs3. Similarly, we observe no immune response in the fetal liver, which instead displays metabolic changes, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. The maternal liver and plasma display similar metabolic alterations, potentially increasing bioavailability of docosahexaenoic acid for the mother and fetus. Thus, our integrated temporal analysis shows that systemic inflammation in the mother leads to a metabolic perturbation in the fetus. Maternal immune activation during pregnancy can negatively impact the developing fetus. Here, applying multi-omics (RNA-seq, phosphoproteomics and lipidomics) and imaging, the authors show that while maternal immune activation induces strong innate response in maternal organs it does not extend through the placenta but leads to fetal metabolic changes.

The tumor microenvironment (TME) is increasingly appreciated to play a decisive role in cancer development and response to therapy in all solid tumors. Hypoxia, acidosis, high interstitial pressure, nutrient-poor conditions, and high cellular heterogeneity of the TME arise from interactions between cancer cells and their environment. These properties, in turn, play key roles in the aggressiveness and therapy resistance of the disease, through complex reciprocal interactions between the cancer cell genotype and phenotype, and the physicochemical and cellular environment. Understanding this complexity requires the combination of sophisticated cancer models and high-resolution analysis tools. Models must allow both control and analysis of cellular and acellular TME properties, and analyses must be able to capture the complexity at high depth and spatial resolution. Here, we review the advantages and limitations of key models and methods in order to guide further TME research and outline future challenges.

Despite the physiological and pathophysiological significance of microenvironmental gradients, e.g., for diseases such as cancer, tools for generating such gradients and analyzing their impact are lacking. Here, we present an integrated microfluidic-based workflow that mimics extracellular pH gradients characteristic of solid tumors while enabling high-resolution live imaging of, e.g., cell motility and chemotaxis, and preserving the capacity to capture the spatial transcriptome. Our microfluidic device generates a pH gradient that can be rapidly controlled to mimic spatiotemporal microenvironmental changes over cancer cells embedded in a 3D matrix. The device can be reopened allowing immunofluorescence analysis of selected phenotypes, as well as the transfer of cells and matrix to a Visium slide for spatially resolved analysis of transcriptional changes across the pH gradient. This workflow is easily adaptable to other gradients and multiple cell types and can therefore prove invaluable for integrated analysis of roles of microenvironmental gradients in biology.

Background and Aims:The regenerative capacity of the pancreas diminishes with age. Understanding acinar cell responses to injury and the resolution of regenerative processes is crucial for tissue homeostasis. However, knowledge about the impact of aging on these processes remains limited. Methods:To investigate the influence of aging on pancreas regeneration, we established a cohort of young (7-14 weeks) and old (18 months) C57bl/6 mice. Experimental pancreatitis was induced using caerulein, and pancreas samples were collected at various time points after induction, covering acute damage response, inflammation, peak proliferation, and inflammation resolution. Our analysis involved immunohistochemistry, quantitative imaging, and gene expression analyses. Results:Our study revealed a significant decline in the regenerative capacity of the pancreas in old mice. Despite similar morphology and transcriptional profiles between the pancreas of young and old mice under homeostasis, the aged pancreas is primed to generate an exacerbated proinflammatory reaction in response to injury. Specifically, we observed notable upregulation of Junb expression in acinar cells and aberrant myofibroblast activation in the aged pancreas. Conclusion:The response of acinar cells to injury in the pancreas of aged mice is characterized by an increased susceptibility to inflammation and stromal reactions. Our findings uncover a pre-existing proinflammatory state in aged acinar cells, offering insights into potential strategies to prevent the onset of pancreatic insufficiency and the development of inflammatory conditions. These insights hold implications for preventing conditions such as chronic pancreatitis and pancreatic ductal adenocarcinoma.

JASPAR (https://jaspar.elixir.no/) is a widely-used open-access database presenting manually curated high-quality and non-redundant DNA-binding profiles for transcription factors (TFs) across taxa. In this 10th release and 20th-anniversary update, the CORE collection has expanded with 329 new profiles. We updated three existing profiles and provided orthogonal support for 72 profiles from the previous release's UNVALIDATED collection. Altogether, the JASPAR 2024 update provides a 20% increase in CORE profiles from the previous release. A trimming algorithm enhanced profiles by removing low information content flanking base pairs, which were likely uninformative (within the capacity of the PFM models) for TFBS predictions and modelling TF-DNA interactions. This release includes enhanced metadata, featuring a refined classification for plant TFs' structural DNA-binding domains. The new JASPAR collections prompt updates to the genomic tracks of predicted TF binding sites (TFBSs) in 8 organisms, with human and mouse tracks available as native tracks in the UCSC Genome browser. All data are available through the JASPAR web interface and programmatically through its API and the updated Bioconductor and pyJASPAR packages. Finally, a new TFBS extraction tool enables users to retrieve predicted JASPAR TFBSs intersecting their genomic regions of interest.

Harsh environments in poorly perfused tumor regions may select for traits driving cancer aggressiveness. Here, we investigated whether tumor acidosis interacts with driver mutations to exacerbate cancer hallmarks. We adapted mouse organoids from normal pancreatic duct (mN10) and early pancreatic cancer (mP4, KRAS-G12D mutation, ± p53 knockout) from extracellular pH 7.4 to 6.7, representing acidic niches. Viability was increased by acid adaptation, a pattern most apparent in wild-type (WT) p53 organoids, and exacerbated upon return to pH 7.4. This led to increased survival of acid-adapted organoids treated with gemcitabine and/or erlotinib, and, in WT p53 organoids, acid-induced attenuation of drug effects. New genetic variants became dominant during adaptation, yet they were unlikely to be its main drivers. Transcriptional changes induced by acid and drug adaptation differed overall, but acid adaptation increased the expression of gemcitabine resistance genes. Thus, adaptation to acidosis increases cancer cell viability after chemotherapy.

Pan-cancer Isoform Switches with Predicted Consequences.

Analysis of the effect of potential biases in TCGA data.

ChIA-PET associations between differentially expressed enhancerRNAs (eRNAs) and promoters of annotated genes.

Overview of literature derived isoform switches as well as the extensions we make.

ABSTRACT Maternal immune system activation (MIA) during pregnancy can disrupt the fetal environment, causing postnatal susceptibility to disorders. How the placenta and the fetus respond to acute MIA over time is unknown. Here, we characterized the response to acute maternal pulmonary inflammation across time in maternal and fetal organs using multi-omics. Unlike maternal organs which mounted strong innate immune responses, the placenta upregulated tissue-integrity genes, likely to prevent fetal exposure to infections, and downregulated growth-associated genes. Subsequently, the placenta upregulated biosynthesis and endoplasmic reticulum stress genes in order to return to homeostasis. These responses likely protected the fetus, since we observed no immune response in fetal liver. Instead, likely due to nutrient depletion, the fetal liver displayed metabolic adaptations, including increases in lipids containing docosahexaenoic acid, crucial for fetal brain development. Our study shows, for the first time, the integrated temporal response to pulmonary MIA across maternal and fetal organs.

List of isoform switches with predicted consequences.