bims-gerecp Biomed News
on Gene regulatory networks of epithelial cell plasticity
Issue of 2025–11–30
nineteen papers selected by
Xiao Qin, University of Oxford
  1. Leveraging WNT Hyperactivation to Kill Colorectal Cancer While Rejuvenating Healthy Intestine.
  2. Functional interrogation uncovers a critical role for a high-plasticity cell state in lung adenocarcinoma.
  3. Lgr5+ Stem Cells Maintain Apex Position in Cell Hierarchy of the Intestinal Epithelium During Homeostasis and Injury.
  4. Polyclonal origins of human premalignant colorectal lesions.
  5. Cell state plasticity emerging from co-regulated, competitive, and configurable interactions within the AP-1 network.
  6. Single-cell insights into tumor microenvironment heterogeneity and plasticity: transforming precision therapy in gastrointestinal cancers.
  7. Precision Oncology: Current Landscape, Emerging Trends, Challenges, and Future Perspectives.
  8. Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage conversion.
  9. A niche-dependent redox rheostat regulates epithelial stem cell fate.
  10. Attractors are less stable than their basins: Canalization creates a coherence gap in gene regulatory networks.
  11. AI and human expertise in cancer care - striving for synergy.
  12. MAPK-driven epithelial cell plasticity drives colorectal cancer therapeutic resistance.
  13. Passenger mutations link cellular origin and transcriptional identity in human lung adenocarcinomas.
  14. Fate and state transitions during human blood vessel organoid development.
  15. Gastric Organoid-Based Ectopic and Orthotopic In Vivo CRISPR Screening for Tumor Suppressors in Gastric Cancer.
  16. Age distinguishes selection from causation in cancer genomes.
  17. Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.
  18. Integrative analysis of genomic and transcriptomic data informs precancer progression in the pancreas.
  19. RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution.
  1. bioRxiv. 2025 Oct 06. pii: 2025.10.05.680591. [Epub ahead of print]
    Leveraging WNT Hyperactivation to Kill Colorectal Cancer While Rejuvenating Healthy Intestine.
    George Eng, Jonathan Braverman, Manish Gala, Omer Yilmaz.
      WNT signaling maintains intestinal homeostasis yet drives colorectal cancer (CRC) when constitutively activated by APC mutations. We overturn the fundamental assumption that APC-mutant tumors exist at maximal WNT activation, revealing instead that cancer cells occupy a precarious "WNT-just-right" zone along a signaling continuum. This discovery exposes an unprecedented therapeutic vulnerability: while normal intestinal epithelium thrives with enhanced WNT signaling, APC-mutant tumor cells undergo apoptosis when pushed beyond their oncogenic setpoint, a phenomenon we term "over-WNTing." Through systematic organoid-based screening, we identified that WNT hyperactivation through multiple approaches: GSK3 inhibition, concentrated WNT proteins, or APC knockdown in GSK3-null backgrounds, selectively kills tumor cells by hyperactivating the driving pathway of CRC. Mechanistically, over-WNTing in APC-mutant cells triggers spillover into non-canonical planar cell polarity signaling, where RHOC upregulation induces ROCK1/2-mediated apoptosis. We demonstrate therapeutic efficacy across the neoplastic continuum, from adenomas to metastatic CRC, including patient-derived tumors, validating GSK3 inhibition with a novel nanoparticle formulation. This discovery enables the first cancer therapy that simultaneously enhances normal tissue function while eliminating tumors. "Over-WNTing" effectively treats adenomas and both mouse and patient-derived CRC, establishing a therapeutic paradigm that exploits fundamental differences in cellular WNT biology to achieve the dual benefit of eliminating cancer while promoting healthy tissue regeneration.
    DOI:  https://doi.org/10.1101/2025.10.05.680591
  2. bioRxiv. 2025 Oct 06. pii: 2025.10.03.680232. [Epub ahead of print]
    Functional interrogation uncovers a critical role for a high-plasticity cell state in lung adenocarcinoma.
    Jason E Chan, Chun-Hao Pan, Jonathan Rub, Klavdija Krause, Emma Brown, Gary Guzman, Zeda Zhang, Hannah Styers, Griffin Hartmann, Zhuxuan Li, Xueqian Zhuang, Scott W Lowe, Doron Betel, Yan Yan, Tuomas Tammela.
      Plasticity-the ability of cells to undergo phenotypic transitions-drives cancer progression and therapy resistance 1-3 . To date, strategies targeting cancer plasticity have not advanced to the clinic due to a lack of fundamental understanding of the underlying mechanisms. Recent studies have suggested that plasticity in solid tumors is concentrated in a minority subset of cancer cells 4-6 , yet functional studies interrogating this high plasticity cell state (HPCS) in situ are lacking. Here, we developed mouse models enabling detection, longitudinal lineage tracing, and ablation of the HPCS in autochthonous lung tumors in vivo . Using lineage tracing, we uncover the HPCS cells are dedifferentiated but possess high capacity for cell state transitions, giving rise to both early neoplastic (differentiated) and advanced lung cancer cell states in situ . Longitudinal lineage tracing using secreted luciferases reveals HPCS-derived cells harbor high capacity for growth when compared to bulk cancer cells or another defined cancer cell state with features of differentiated lung epithelium. Suicide gene-mediated ablation of the HPCS in early neoplasias abrogates tumor progression. Ablating HPCS cells in established tumors by suicide gene or HPCS-directed CAR T cells robustly reduces tumor burden, whereas ablation of a differentiated lung cancer cell state had no effect. We further demonstrate that the HPCS gives rise to therapy-resistant cell states, whereas ablation of the HPCS abrogates resistance to chemotherapy and oncoprotein-targeted therapy. Interestingly, an HPCS-like state is ubiquitous in regenerating epithelia and in carcinomas of multiple other tissues, revealing a convergence of plasticity programs. Our work establishes the HPCS as a critical hub enabling reciprocal transitions between cancer cell states, including acquisition of states adapted to cancer therapies. Targeting the HPCS in lung cancer and in other carcinomas may suppress cancer progression and eradicate treatment resistance.
    DOI:  https://doi.org/10.1101/2025.10.03.680232
  3. bioRxiv. 2025 Nov 11. pii: 2025.11.09.687498. [Epub ahead of print]
    Lgr5+ Stem Cells Maintain Apex Position in Cell Hierarchy of the Intestinal Epithelium During Homeostasis and Injury.
    Julian Chua, Lucy Driver, Masahiro Narimatsu, Mardi Fink, Lixia Luo, Jordan Tran, Arshdeep Kaur, Rida Habib, Jatin Roper, David G Kirsch, Jeffrey L Wrana, Chang-Lung Lee, Arshad Ayyaz.
      The cellular origin of intestinal epithelial homeostasis and regeneration has been a subject of continued debate, with recent models challenging the primacy of WNT-dependent Lgr5⁺ crypt base columnar (CBC) cells as the central intestinal stem cell population. Here, we revisit this question through quantitative integration of single-cell transcriptomic, chromatin accessibility, spatial, and lineage-tracing analyses across the proximal-to-distal axis of the small intestinal epithelium. Our data show that under homeostatic conditions, Lgr5⁺ cells exclusively sustain epithelial self-renewal in nearly all crypt-villus units along the entire length of the small intestine, a process for which R-spondin is indispensable. Following irradiation or chemotoxic injury, surviving Lgr5⁺ cells and their progeny reprogram into transient fetal-like cell states that initiate epithelial repair. Crucially, successful regeneration depends on the reactivation of canonical WNT/β-catenin signaling, as evidenced by increased TCF motif accessibility and upregulation of WNT target genes in newly forming Lgr5 + stem cells. Accordingly, pharmacological inhibition of WNT signaling blocks the reconstitution of Lgr5⁺ cells and crypt regeneration, leading to epithelial collapse. These findings reconcile prior controversies by demonstrating the central role of Lgr5⁺ CBC cells in epithelial self-renewal and regeneration following injury.
    DOI:  https://doi.org/10.1101/2025.11.09.687498
  4. Nature. 2025 Nov 25.
    Polyclonal origins of human premalignant colorectal lesions.
    Debra Van Egeren, Ryan O Schenck, Aziz Khan, Aaron M Horning, Shanlan Mo, Clemens L Weiß, Edward D Esplin, Winston R Becker, Si Wu, Casey Hanson, Nasim Barapour, Lihua Jiang, Kévin Contrepois, Hayan Lee, Stephanie A Nevins, Tuhin K Guha, Hao Zhang, Zhen He, Zhicheng Ma, Emma Monte, Thomas V Karathanos, Rozelle Laquindanum, Meredith A Mills, Hassan Chaib, Roxanne Chiu, Ruiqi Jian, Joanne Chan, Mathew Ellenberger, Bahareh Bahmani, Basil Michael, Annika K Weimer, D Glen Esplin, Samuel Lancaster, Jeanne Shen, Uri Ladabaum, Teri A Longacre, Anshul Kundaje, William J Greenleaf, Zheng Hu, James M Ford, Michael P Snyder, Christina Curtis.
      Cancer is generally thought to be caused by expansion of a single mutant cell1. However, analyses of early colorectal cancer lesions suggest that tumors may instead originate from multiple, genetically distinct cell populations2,3. Detecting polyclonal tumor initiation is challenging in patients, as it requires profiling early-stage lesions before clonal sweeps obscure diversity. To investigate this, we analyzed normal colorectal mucosa, benign and dysplastic premalignant polyps, and malignant adenocarcinomas (123 samples) from six individuals with familial adenomatous polyposis (FAP). Individuals with FAP have a germline heterozygous APC mutation, predisposing them to colorectal cancer and numerous premalignant polyps by early adulthood4. Whole-genome and/or whole-exome sequencing revealed that many premalignant polyps-40% with benign histology and 28% with dysplasia-were composed of multiple genetic lineages that diverged early, consistent with polyclonal origins. This conclusion was reinforced by whole-genome sequencing of single crypts from multiple polyps in additional patients which showed limited sharing of mutations among crypts within the same lesion. In some cases, multiple distinct APC mutations co-existed in different lineages of a single polyp, consistent with polyclonality. These findings reshape our understanding of early neoplastic events, demonstrating that tumor initiation can arise from the convergence of diverse mutant clones. They also suggest that cell-intrinsic growth advantages alone may not fully explain tumor initiation, highlighting the importance of microenvironmental and tissue-level factors in early cancer evolution.
    DOI:  https://doi.org/10.1038/s41586-025-09930-y
  5. bioRxiv. 2025 Nov 07. pii: 2025.11.06.686987. [Epub ahead of print]
    Cell state plasticity emerging from co-regulated, competitive, and configurable interactions within the AP-1 network.
    Yonatan N Degefu, Magda Bujnowska, Douglas G Baumann, Mohammad Fallahi-Sichani.
      Cell state plasticity drives metastasis and therapy resistance in cancers. In melanoma, these behaviors map onto a melanocytic-to-mesenchymal-like continuum regulated by AP-1 transcription factors. However, how the AP-1 network encodes a limited set of discrete states, why their distributions vary across tumors, and what drives phenotypically consequential AP-1 state transitions remain unclear. We develop a mechanistic ODE model of the AP-1 network capturing their dimerization-controlled, co-regulated, competitive interactions. Calibrated to heterogeneous single-cell data across genetically diverse melanoma populations and combined with statistical learning, the model reveals network features explaining population-specific AP-1 state distributions. These features correlate with MAPK activity across tumor lines and with variability within clones, linking MAPK signaling to AP-1 states. The model predicts and experiments validate adaptive AP-1 reconfiguration under MAPK inhibition, inducing a dedifferentiated, therapy-resistant state that can be blocked by model-guided AP-1 perturbations. These results establish AP-1 as a configurable network and provide a computational framework for predicting and modulating AP-1 driven cell state plasticity.
    DOI:  https://doi.org/10.1101/2025.11.06.686987
  6. J Exp Clin Cancer Res. 2025 Nov 28. 44(1): 314
    Single-cell insights into tumor microenvironment heterogeneity and plasticity: transforming precision therapy in gastrointestinal cancers.
    Jialei Weng, Feng Ju, Zicheng Lyu, Ningbo Fan, Daniel J Smit, Wenxin Xu, Xiaolin Wu, Philip Becker, Yinan Xu, Michal R Schweiger, Axel M Hillmer, Ralf Harwig, Sheraz Gul, Alexander Link, Lydia Meder, Nan Fang, Qiongzhu Dong, Christiane J Bruns, Ning Ren, Yue Zhao.
      The development and progression of gastrointestinal (GI) cancers not only depend on the malignancy of the tumor cells, but is also defined by the complex and adaptive nature of the tumor microenvironment (TME). The TME in GI cancers exhibits a complex internal structure, typically comprising cancer cells, cancer stem cells, cancer-associated fibroblasts, immune cells, and endothelial cells, all embedded within a dynamic extracellular matrix. This intricate ecosystem fuels tumor initiation, progression, metastasis, recurrence and therapy response through the heterogeneity and plasticity. Recent advances in single-cell sequencing have provided unprecedented resolution in profiling the cellular diversity and interactions within the TME. These technologies have uncovered previously unknown cell subtypes and intricate communication networks that drive therapy resistance and tumor relapse. In this review, we summarize and discuss the latest findings from single-cell sequencing of key cellular players and their interactions within the TME of GI cancers. We highlight single cell insights that are reshaping our understanding of tumor biology, with particular focus on their implications for overcoming therapy resistance and improving clinical outcomes. We believe that a deeper understanding of TME heterogeneity and plasticity at the single-cell level promises to transform the landscape of precision treatment in GI cancers.
    Keywords:  Cancer-associated fibroblasts; Cell subsets; Gastrointestinal cancers; Heterogeneity; Plasticity; Single-cell RNA sequencing; Tumor microenvironment
    DOI:  https://doi.org/10.1186/s13046-025-03567-5
  7. Cells. 2025 Nov 17. pii: 1804. [Epub ahead of print]14(22):
    Precision Oncology: Current Landscape, Emerging Trends, Challenges, and Future Perspectives.
    Diane Qiao, Richard C Wang, Zhixiang Wang.
      Precision oncology is broadly defined as cancer prevention, diagnosis, and treatment specifically tailored to the patient based on his/her genetics and molecular profile. In simple terms, the goal of precision medicine is to deliver the right cancer treatment to the right patient, at the right dose, at the right time. Precision oncology is the most studied and widely applied subarea of precision medicine. Now, precision oncology has expanded to include modern technology (big data, single-cell spatial multiomics, molecular imaging, liquid biopsy, CRISPR gene editing, stem cells, organoids), a deeper understanding of cancer biology (driver cancer genes, single nucleotide polymorphism, cancer initiation, intratumor heterogeneity, tumor microenvironment ecosystem, pan-cancer), cancer stratification (subtyping of traditionally defined cancer types and pan-cancer re-classification based on shared properties across traditionally defined cancer types), clinical applications (cancer prevention, early detection, diagnosis, targeted therapy, minimal residual disease monitoring, managing drug resistance), lifestyle changes (physical activity, smoking, alcohol consumption, sunscreen), cost management, public policy, and more. Despite being the most developed area in precision medicine, precision oncology is still in its early stages and faces multiple challenges that need to be overcome for its successful implementation. In this review, we examine the history, development, and future directions of precision oncology by focusing on emerging technology, novel concepts and principles, molecular cancer stratification, and clinical applications.
    Keywords:  cancer initiation; liquid biopsy; multiomics; precision medicine; precision oncology; single-cell spatial omics; stratification; subtyping; targeted therapy; tumor heterogeneity; tumor microenvironment
    DOI:  https://doi.org/10.3390/cells14221804
  8. Nat Commun. 2025 Nov 27.
    Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage conversion.
    Maria T Bejar, Paula Jimenez-Gomez, Ilias Moutsopoulos, Harikrishnan Ajith, Bartomeu Colom, Jamie McGinn, Seungmin Han, Greta Skrupskelyte, Fernando J Calero-Nieto, Berthold Göttgens, Irina I Mohorianu, Benjamin D Simons, Maria P Alcolea.
      The ability of adult epithelial cells to rewire their cell fate programme in response to injury has emerged as a new paradigm in stem cell biology. This plasticity supersedes the concept of strict stem cell hierarchies, granting cells access to a wider repertoire of fate choices. Yet, in order to prevent a disordered cellular response, this process must be finely regulated. Here we investigate the little-known regulatory processes that restrict fate permissibility in adult cells, and keep plasticity in check. Using a 3D regenerative culture system, that enables co-culturing epithelium and stroma of different origins, we demonstrate that oesophageal cells exposed to the ectopic signals of the dermis are capable of switching their identity towards skin. Lineage tracing experiments and histological analysis, however, reveal that the oesophageal-to-skin lineage conversion process is highly inefficient, pointing to the existence of barriers limiting cell fate re-specification. Single-cell RNA sequencing capturing the temporality of this process shows that cells transitioning towards skin identity resist the natural progression towards tissue maturation by remaining in a persistent regenerative state marked by a particularly strong hypoxic signature. Gain and loss of function experiments demonstrate that the HIF1a-SOX9 axis acts as a key modulator of epithelial cell fate plasticity, restricting changes in identity during tissue regeneration. Taken together, our results reveal the existence of lineage conversion barriers that must be resolved for cells to respond to signals instructing alternative fate choices, shedding light on the principles underlying the full regenerative capacity of adult epithelial cells.
    DOI:  https://doi.org/10.1038/s41467-025-66446-9
  9. Nat Commun. 2025 Nov 28.
    A niche-dependent redox rheostat regulates epithelial stem cell fate.
    Xi Chen, Krishnan Raghunathan, Bin Bao, Elsy Ngwa, Andrew Kwong, Zhongyang Wu, Stephen Babcock, Clara Baek, George Ye, Anoohya Muppirala, Qianni Peng, Michael Rutlin, Mantu Bhaumik, Daping Yang, Daniel Kotlarz, Unmesh Jadhav, Meenakshi Rao, Eranthie Weerapana, Xu Zhou, Jose Ordovas-Montanes, Scott B Snapper, Jay R Thiagarajah.
      Intestinal stem cells (ISCs) reside in regionally variable niches that provide diverse microenvironmental cues such as tissue oxygen status, and morphogen signaling. Integration of these cues with ISC metabolism and fate remains poorly understood. Here, we show that cellular redox balance orchestrates niche factors with metabolic state to govern cell fate decisions. We demonstrate that hypoxia and Wnt signaling synergistically restrict the reactive oxygen species generating enzyme NADPH oxidase 1 (NOX1) regionally to the crypt base in the distal colon. NOX1 enables maintenance of an oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric ISC self-renewal and lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1α-dependent signaling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1. Our studies show redox balance acts as a cellular rheostat that is central and causative for metabolic control of the ISC cell-cycle.
    DOI:  https://doi.org/10.1038/s41467-025-66636-5
  10. bioRxiv. 2025 Nov 08. pii: 2025.11.06.687062. [Epub ahead of print]
    Attractors are less stable than their basins: Canalization creates a coherence gap in gene regulatory networks.
    Venkata Sai Narayana Bavisetty, Matthew Wheeler, Claus Kadelka.
      Waddington's epigenetic landscape has served as biology's central metaphor for cellular differentiation for over half a century, depicting mature cell types as balls resting in stable valley floors. Boolean networks - introduced by Kauffman in 1969 to model gene regulatory dynamics - provide a mathematical formalization of this landscape, where attractors represent phenotypes and basins of attraction correspond to developmental valleys. Traditional stability measures quantify robustness by perturbing arbitrary states, yet biological systems typically reside at attractors rather than in transient states. Here we formalize and systematically analyze attractor coherence - a stability measure Kauffman originally envisioned but never rigorously developed - which quantifies how likely a perturbation of an attractor state causes phenotype switching. Analyzing 122 expertcurated biological Boolean models, we reveal a striking paradox: attractors representing mature cell types are consistently less stable than the developmental trajectories approaching them. Largescale simulations of random networks demonstrate that this coherence gap arises from canalization - a hallmark of biological regulation where individual genes can override others. While canalization increases overall network stability, it disproportionately stabilizes transient states, positioning attractors near basin boundaries. The gap's magnitude is almost perfectly predicted by network bias (Spearman's ρ = -0.997), itself modulated by canalization. These findings revise Waddington's landscape: canalization carves deep protective valleys ensuring developmental robustness, yet simultaneously flattens ridges near valley floors, facilitating phenotypic plasticity when multiple fates coexist. This explains how biological systems achieve both reliable development and plasticity, with implications for understanding development, disease-related transitions, and designing robust yet controllable synthetic gene circuits.
    DOI:  https://doi.org/10.1101/2025.11.06.687062
  11. Nat Rev Clin Oncol. 2025 Nov 25.
    AI and human expertise in cancer care - striving for synergy.
    Milit S Patel, Edward Christopher Dee, Nancy Y Lee.
      
    DOI:  https://doi.org/10.1038/s41571-025-01108-9
  12. Nature. 2025 Nov 24.
    MAPK-driven epithelial cell plasticity drives colorectal cancer therapeutic resistance.
    Mark White, Megan L Mills, Laura M Millett, Kathryn Gilroy, Yourae Hong, Lucas B Zeiger, Rosalin J Simpson, Shania M Corry, Amelia Ligeza, Tamsin R M Lannagan, Susanti Susanti, Rachel A Ridgway, Ayse S Yazgili, Lucile Grzesiak, Raheleh Amirkhah, Catriona A Ford, Nikola Vlahov, Hannah Tovell, Leah Officer-Jones, Catherine Ficken, Rachel Pennie, Arafath K Najumudeen, Alexander Raven, Nadia Nasreddin, Ekansh Chauhan, Andrew S Papanastasiou, Colin Nixon, Vivienne Morrison, Rene Jackstadt, Janet S Graham, Crispin J Miller, Sarah J Ross, Simon T Barry, Valeria Pavet, Richard H Wilson, John Le Quesne, Philip D Dunne, Sabine Tejpar, Simon Leedham, Andrew D Campbell, Owen J Sansom.
      The colorectal epithelium is rapidly renewing, with remarkable capacity to regenerate following injury. In colorectal cancer (CRC), this regenerative capacity can be co-opted to drive epithelial plasticity. While oncogenic MAPK signalling in CRC is common, with frequent mutations of both KRAS (40-50%) and BRAF (10%)1, inhibition of this pathway typically drives resistance clinically. Given the development of KRAS inhibitors, and licensing of BRAF inhibitor combinations2-4, we have interrogated key mechanisms of resistance to these agents in advanced preclinical CRC models. We show that oncogenic MAPK signalling induces epithelial state changes in vivo, driving adoption of a regenerative/revival stem like population, while inhibition leads to rapid transcriptional remodeling of both Kras- and Braf-mutant tumours, favoring a Wnt-associated, canonical stem phenotype. This drives acute therapeutic resistance in Kras- and delayed resistance in Braf-driven models. Importantly, where plasticity is restrained, such as in early metastatic disease, or through targeting ligand-dependent Wnt-pathway Rnf43 mutations, marked therapeutic responses are observed. This explains the super response to BRAF+EGFR targeted therapies previously observed in a BRAF/RNF43 co-mutant patient population, highlighting the criticality of cellular plasticity in therapeutic response. Together, our data provides clear insight into the mechanisms underpinning resistance to MAPK targeted therapies in CRC. Moreover, strategies that aim to corral stem cell fate, restrict epithelial plasticity or intervene when tumours lack heterogeneity may improve therapeutic efficacy of these agents.
    DOI:  https://doi.org/10.1038/s41586-025-09916-w
  13. Nat Genet. 2025 Nov 27.
    Passenger mutations link cellular origin and transcriptional identity in human lung adenocarcinomas.
    Sukanya Panja, Padmaja Mantri, Kofi Ennu Johnson, Juan Sebastian Andrade-Martinez, Soo-Ryum Yang, Aditya Deshpande, Huasong Tian, Shaham Beg, Kentaro Ohara, Alessandro Leal, Joel Rosiene, Marlon Stoeckius, Peter Smibert, William D Travis, Juan Miguel Mosquera, Paz Polak, Marcin Imieliński.
      DNA damage is preferentially repaired in expressed genes; thus, genome-wide correlations between somatic mutation patterns and normal cell transcription may reflect tumor cell origins. Accordingly, we found that aggregate lung adenocarcinoma (LUAD) and squamous cancer (LUSC) somatic mutation density associated most strongly with distal (alveolar) and proximal (basal) lung cell-type-specific gene expression, respectively, consistent with presumed LUAD and LUSC cell origins. Analyzing individual genomes, 21% of LUADs bore mutational footprints of proximal airway origins, with 38% classified as ambiguous. Distal origin LUADs, enriched for KRAS and STK11 drivers, occurred mainly in smokers; proximal origin LUADs, enriched for EGFR drivers, were more common in never-smokers. Ambiguous origin LUADs showed APOBEC signatures and SMARCA4 alterations. TP53 mutant LUADs with non-distal cell origins preferentially exhibited non-distal transcriptional identity. Our study reveals a complex interplay between lineage and identity in LUAD evolution and offers a scalable strategy to infer tumor origins in human cancers.
    DOI:  https://doi.org/10.1038/s41588-025-02418-5
  14. Cell. 2025 Nov 21. pii: S0092-8674(25)01314-5. [Epub ahead of print]
    Fate and state transitions during human blood vessel organoid development.
    Marina T Nikolova, Zhisong He, Makiko Seimiya, Gustav Jonsson, Wuji Cao, Ryo Okuda, Reiner A Wimmer, Ryoko Okamoto, Jonas M Nikoloff, Petra S Dittrich, Josef M Penninger, J Gray Camp, Barbara Treutlein.
      
    DOI:  https://doi.org/10.1016/j.cell.2025.11.018
  15. Gastroenterology. 2025 Nov 25. pii: S0016-5085(25)05992-X. [Epub ahead of print]
    Gastric Organoid-Based Ectopic and Orthotopic In Vivo CRISPR Screening for Tumor Suppressors in Gastric Cancer.
    Jiazhuo He, Giovanni Papa, Flora Azizi, Lydia Kirsche, Mariela Artola-Boran, Rafaela Ferreira Cassio, Adriana Lea Hotz, Gavin Geiger, Biel Francas, Achim Weber, Alexandar Tzankov, Zacharias Kontarakis, Peter Leary, Anne Müller.
       BACKGROUND & AIMS: CRISPR-Cas9 screening is a powerful tool for the in vivo discovery of cancer dependencies. The aim of this study was to perform in vivo CRISPR knockout screening for gastric tumor suppressors using gastric murine organoids in a subcutaneous as well as a surgical model of orthotopic tumor growth.
    METHODS: In vivo screening was performed using a custom library targeting 49 putative gastric tumor suppressor genes, as well as a "cancer genome-wide" library targeting 5000 genes, in immunocompetent and -deficient mice, and in the presence or absence of the gastric pathogen Helicobacter pylori. The top hits were selected for individual validation and mechanistic follow-up.
    RESULTS: Our custom library knockout screens revealed single-guide RNAs targeting Pten, Fbxw7, and genes encoding several components of the transforming growth factor-ß signaling pathway (Smad4, Tgfbr1, Tgfbr2, and Acvr2a) to be recurrently enriched both in subcutaneously and orthotopically growing tumors. The same, and several additional genes were identified by cancer genome-wide CRISPR screening. Ten of our top hits could be validated individually in vivo. Pten inactivation resulted in large tumors characterized by increased neo-angiogenesis, neutrophil recruitment, and T-cell exclusion. Inactivation of Smad4, Tgfbr1, or Acvr2a all produced phenotypes that were reminiscent of early gastric cancer precursor lesions such as intestinal Alcian blue-positive metaplasia and compensatory hyperplasia. Helicobacter pylori infection failed to affect the mutational landscape of tumors; rather, we found that H pylori modulates the tumor microenvironment and recruits large numbers of tumor-promoting SiglecF+ neutrophils.
    CONCLUSIONS: In summary, we describe here a versatile model of gastric carcinogenesis that uncouples the genetics of the tumor and the host, and that faithfully recapitulates key risk factors of the malignancy.
    Keywords:  In Vivo CRISPR Knockout Screen; Orthotopic Modeling of Gastric Carcinogenesis; Tumor Microenvironment; Tumor Suppressor Genes; Tumor-Promoting Neutrophils
    DOI:  https://doi.org/10.1053/j.gastro.2025.09.009
  16. bioRxiv. 2025 Oct 06. pii: 2025.10.06.680730. [Epub ahead of print]
    Age distinguishes selection from causation in cancer genomes.
    David Cheek, Martin Blohmer, Martin Nowak, Tibor Antal, Kamila Naxerova.
      Cancer genome sequencing efforts have revealed hundreds of genes under positive selection, many of which are now being developed as therapeutic targets. However, positively selected mutations also populate our aging tissues in the absence of cancer. For most mutations, it is currently unknown whether they are recurrently found in cancer genomes because they cause cancer or because they expand during normal tissue evolution and are passively inherited. Here, we develop a mathematical and statistical framework that distinguishes these two factors. We discover - across thousands of cancer and normal tissue genomes - that mutations that most strongly increase cancer risk are enriched in younger patients' cancers, whereas mutations that are positively selected in normal tissue without causing cancer are enriched in older patients. Focusing on a particularly data-rich cancer type, acute myeloid leukemia, we show that genetic differences between young- and adult-onset cancers can largely be explained by the cumulative effects of normal tissue evolution, contradicting the long-standing notion that childhood cancers require a distinct set of causal mutations. Our framework establishes patient age as a powerful resource for clarifying whether positively selected mutations in cancer genomes are truly disease-promoting.
    DOI:  https://doi.org/10.1101/2025.10.06.680730
  17. Nat Methods. 2025 Nov 24.
    Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.
    Xin Sui, Jennifer A Lo, Shuchen Luo, Yichun He, Zefang Tang, Zuwan Lin, Dániel L Barabási, Yiming Zhou, Wendy Xueyi Wang, Jia Liu, Xiao Wang.
      Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 µm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 60-200-µm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.
    DOI:  https://doi.org/10.1038/s41592-025-02867-0
  18. bioRxiv. 2025 Nov 04. pii: 2025.11.03.686234. [Epub ahead of print]
    Integrative analysis of genomic and transcriptomic data informs precancer progression in the pancreas.
    Kathleen Noller, Jiaying Lai, Daniel Lesperance, Ricky S Adkins, Ahmed Elhossiny, Paola A Guerrero, Kimal I Rajapakshe, Anirban Maitra, Michelle Giglio, Anup Mahurkar, Owen White, Marina Pasca Di Magliano, Michael F Ochs, Luciane T Kagohara, Laura D Wood, Rachel Karchin, Elana J Fertig.
      Pancreatic ductal adenocarcinoma (PDAC) arises from heterogeneous precursor lesions, including intraductal papillary mucinous neoplasms (IPMNs), but the features distinguishing indolent from progressive lesions remain unclear. We performed an integrative analysis of transcriptomic, genomic, and microenvironmental profiles of IPMNs to define multi-omic phenotypes. Using transfer learning, we projected IPMN-derived transcriptional programs onto spatial transcriptomic datasets from IPMNs and pancreatic intraepithelial neoplasias (PanINs). We identified two major phenotypes: one associated with cancer-associated fibroblasts and epithelial-to-mesenchymal transition, shared across IPMN, PanIN, and PDAC; and a second, glycolysis-enriched phenotype with a unique somatic mutation profile specific to IPMN. Spatial mapping further revealed grade-specific enrichment of transcriptional programs and distinct interactions with stromal and immune subtypes, underscoring the role of the precancer microenvironment in progression. These findings establish multi-omic phenotypes that unify genetic, transcriptional, and microenvironmental heterogeneity, providing a framework for distinguishing progressive from indolent precancers and a web-based public atlas for future exploration of these data and transcriptional phenotypes.
    DOI:  https://doi.org/10.1101/2025.11.03.686234
  19. bioRxiv. 2025 Nov 05. pii: 2025.11.04.686676. [Epub ahead of print]
    RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution.
    Ruzbeh Mosadeghi, Daniel Foyt, Louis Sharp, Catherine Taylor, Neil Tay, Stefan Oberlin, John Fan, Struan Bourke, Michael Kattah, Bo Huang, Michael T McManus.
      High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identification in both arrayed and pooled formats. As a proof of concept, we applied RainBar to dissect NF-κB signaling dynamics in epithelial cells. Live imaging of RelA translocation uncovered stimulus-specific kinetics: IL-1β triggered rapid recovery, while TNF induced prolonged nuclear localization. In pooled CRISPRi screens, RainBar recovered known NF-κB regulators (e.g., IL1R1, MYD88, TNFRSF1A) and highlighted additional modulators, including the Ino80 chromatin remodeling complex subunits and KAT2A acetyltransferase. Together, these results position RainBar as a flexible platform for multiplexed, image-based functional genomics, with potential to reveal dynamic signaling architectures across diverse cellular contexts in live cells.
    DOI:  https://doi.org/10.1101/2025.11.04.686676
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