The benign-to-malignant transition is a defining step in cancer progression. To investigate when and how malignancy initiation occurs and tissue reorganization proceeds, we combine single-cell and spatial transcriptomic profiling in mouse models of pancreatic ductal adenocarcinoma (PDAC) that capture spontaneous p53 loss. Among Kras-mutant cells, we find that oncogenic and tumor-suppressive programs, including those controlled by p53, CDKN2A, and SMAD4, are co-activated in a discrete progenitor-like population, engaging senescence-like responses. Using a framework we developed for spatial analysis, we show that a niche centered on these cells undergoes stepwise remodeling during tumor progression, mirroring invasive PDAC. Transient KRAS inhibition depletes progenitor-like cells and dismantles their niche, delaying malignancy initiation. Conversely, p53 suppression enables progenitor cell expansion, epithelial-mesenchymal reprogramming, and immune-privileged niche formation. These findings position the progenitor-like state at the convergence of cancer-driving mutations, plasticity, and tissue remodeling, revealing a critical window for intercepting malignancy.

BACKGROUND & AIMS:Although the majority of human pancreatic cancers are classified as pancreatic ductal adenocarcinomas, tumors are comprised of diverse dedifferentiated tumor cell states that dynamically interact with an equally complex microenvironment, making it imperative to understand, in vivo, how normal epithelial identity is (dys)regulated during tumorigenesis. METHODS:Here, we integrate data from mouse and human pancreatic ductal adenocarcinoma with a unique zebrafish model of pancreatic cancer to elucidate evolutionarily conserved mechanisms regulating dedifferentiation during pancreatic tumorigenesis. RESULTS:Histologic and single-cell transcriptional profiling of heterogeneous tumors developing in dual-transgenic ptf1a:Gal4-VP16; UAS:mKO2- KRASG12D adult zebrafish revealed mixed populations of progenitor-like cells with acinar features, progenitor-like cells with ductal features and undifferentiated progenitor-like cells, providing the opportunity to interrogate specific transcriptional and epigenetic modifiers controlling neoplastic pancreatic cellular differentiation. Screening a panel of chromatin modifiers and chromatin readers for differential expression in these distinct cell clusters revealed hdac9b to be upregulated in the progenitor-like cells enriched for ductal features. RNA velocity trajectories supported an acinar cell-of-origin for these hdac9b-expressing progenitor-like cells. The treatment of fish with the class IIA histone deacetylase inhibitor TMP195 confirmed a functional role of histone deacetylase activity in regulating neoplastic cell differentiation through repression of the ductal-like and progenitor cell states and the associated reactivation of acinar gene expression. Cross-species analyses in autochthonous murine tumors confirmed an early, evolutionarily conserved role for Hdac9 in pancreatic tumorigenesis, marking tumor cell subpopulations characterized by a loss of epithelial differentiation and a basal subtype identity. In human pancreatic cancer, higher HDAC9 expression is significantly correlated with poorly differentiated tumor histology and predicts shorter overall survival in patients with pancreatic ductal adenocarcinoma with classical subtype-enriched tumors. CONCLUSIONS:These findings suggest a highly conserved role for HDAC9 and class IIA histone deacetylases in vertebrate pancreatic tumorigenesis and may lead to new strategies for reactivating (normal acinar-epithelial) differentiation programs to intercept and treat pancreatic ductal adenocarcinoma.

GATA6 and GATA4 play key roles in pancreatic development and are essential to maintain the classical transcriptional program in pancreatic ductal adenocarcinoma (PDAC). Using genetic mouse models we show that, in contrast to GATA6, GATA4 is dispensable for the maintenance of acinar homeostasis in the adult pancreas. Deletion of Gata4 in mice expressing mutant Kras in the embryonic pancreas (KG4C) leads to PDAC development in the absence of tissue remodeling, pancreatic intraepithelial neoplasia (PanIN), or other canonical precursor lesions present in Gata4-proficient (KC) mice. Similar observations were made when Gata4 was selectively inactivated in adult, Kras-mutant, acinar cells. We identify Pale Acinar Lesions (PALes) as a previously unrecognized pancreatic lesion, distinct from acino-ductal metaplasia (ADM) and PanINs, present in KC and KG4C mice but not in wild type mice. PALes display weak expression of acinar and ductal markers and lack mucins; they have lower proliferation rates than PanINs. RNA-seq and ChIP-seq reveal that GATA4 and GATA6 partially share genomic binding sites and transcriptomic effects, but they exert opposing influences on mutant Kras-induced, haematopoietic cell-dependent, transcriptional inflammatory programs. Adenoviral-mediated pancreatic expression of IL17 restored the formation of ductal lesions in KG4C mice but failed to rescue PanIN development. Our data indicate that GATA4 functions through the coordinated action of multiple inflammatory factors that are required for ADM/PanIN formation but are dispensable for PDAC development. Collectively, these findings challenge current paradigms of PDAC initiation and progression.

Supplementary Video S1 from Senescence Rewires Microenvironment Sensing to Facilitate Antitumor Immunity

RNA-Seq data of proliferating (PRO) or senescent (SEN) NSP liver tumor cells, for both p53-restoration and drug-induced (trametinib+palbociclib) settings. PRO and SEN cells were also treated with the BET inhibitor JQ-1 (500 n, 48 h), to expose BRD4-mediated transcriptional output in each cellular state.

Supplementary figures complement main figures to show that senescent cells have a rewired environmental signal sensing phenotype, exemplified by an enhanced IFN-g signaling, to facilitate anti-tumor immunity.

The role of driver gene mutations in sustaining tumor growth at metastatic sites is poorly understood. SMAD4 inactivation is a paradigm of such mutations and a hallmark of pancreatic ductal adenocarcinoma (PDAC). To determine whether metastatic tumors are dependent on SMAD4 inactivation, we developed a mouse model of PDAC that enables spatiotemporal control of Smad4 expression. While Smad4 inactivation in the premalignant pancreas facilitated the formation of primary tumors, Smad4 reactivation in metastatic disease suppressed liver metastases but promoted lung metastases. These divergent effects were underpinned by organ-biased differences in the tumor cells' chromatin state that emerged in the premalignant pancreas and were distinguished by the dominance of KLF4 versus RUNX1 transcription factors. Our results show how epigenetic states favored by the organ of residence can influence the output of driver mutations in metastatic tumors, which has implications for interpreting tumor genetics and therapeutically targeting metastatic disease.

Low (50 pg/ml) and high (1 ng/ml) dose of IFN-γ treatment in proliferating and senescent NSP cells.

Supplementary Video S2 from Senescence Rewires Microenvironment Sensing to Facilitate Antitumor Immunity

The classical view of cancer as a genetically driven disease has been challenged by recent findings of oncogenic mutations in phenotypically healthy tissues, refocusing attention on non-genetic mechanisms of tumor initiation. In this context, gene–environment interactions take the stage, with recent studies showing how they unleash and redirect cellular and tissue plasticity towards protumorigenic states in response to the exposome, the ensemble of environmental factors impinging on tissue homeostasis. We conceptualize tumor-initiating plasticity as a phenotype-transforming force acting at three levels: cell-intrinsic, focusing on mutant epithelial cells’ responses to environmental variation; reprogramming of non-neoplastic cells of the host, leading to protumor micro- and macroenvironments; and microbiome ecosystem dynamics. This perspective highlights cell, tissue, and organismal plasticity mechanisms underlying tumor initiation that are shaped by the exposome, and how their functional investigation may provide new opportunities to prevent, detect, and intercept cancer-promoting plasticity.

The transition from benign to malignant growth is a pivotal yet poorly understood step in cancer progression that marks the shift from a pathologically inert condition to a clinically lethal disease. Here, we integrate lineage tracing, single-cell and spatial transcriptomics to visualize the molecular, cellular and tissue-level events that promote or restrain malignancy during the tumor initiation in mouse models of pancreatic ductal adenocarcinoma (PDAC). We identify a discrete progenitor-like population of KRAS-mutant cells that co-activates oncogenic and tumor-suppressive programs-including p53, CDKN2A, and SMAD4-engaging senescence-like responses and remodeling their microenvironment, ultimately assembling a niche that mirrors invasive PDAC. KRAS inhibition depletes progenitor-like cells and dismantles their niche. Conversely, p53 suppression enables progenitor cell expansion, epithelial-mesenchymal reprogramming, and immune-privileged niche formation. These findings position the progenitor-like state as the convergence point of cancer-driving mutations, plasticity, and tissue remodeling-revealing a critical window for intercepting malignancy at its origin.

Differential expression analyses of CD8 T and macrophages populations of proliferating (p53 off) vs. senescent (p53 on) tumors by scRNA-seq.

Senescence triggers an immune evasion-to-immune recognition tumor switch. A, Representative images of CD45 and GFP staining marking immune cells and tumor cells, respectively, in p53-suppressed and p53-restored tumor (7 days after p53 restoration). Right, the quantification of the area of CD45+ staining calculated from 3 random fields per mouse. Each dot represents a mouse. B, Flow cytometry analysis of the global immune landscape in an orthotopic NSP liver tumor model. Immunophenotyping of senescent tumors is performed 9 days after Dox withdrawal, a time point when the senescent state is fully established, yet preceding the massive tumor regression. G-MDSC, granulocytic myeloid-derived suppressor cells; M-MDSC, monocytic myeloid-derived suppressor cells. Data are pooled from 2 independent experiments, with n = 7 in the proliferating group and n = 9 in the senescent group. Note that, as the absolute number of CD45+ cells increases in senescent NSP tumor lesions (A), so do the total numbers of the indicated cell types. C, Flow cytometry analysis of CD8 T cells. Data are pooled from 2 independent experiments, with n = 11 in the proliferating and n = 10 in the senescent groups. Experiments were performed 9 days after Dox withdrawal. D, Representative tissue clearing images of the orthotopic NSP liver tumors. T cells, neutrophils, and vasculature are labeled by CD3, MPO, and CD31 staining, respectively. Samples were collected 9 days after Dox withdrawal. E, Tumor size change measured by ultrasound upon p53 restoration in mice after depleting specific immune cell types using antibodies or drugs. F, Left, uniform manifold approximation and projection (UMAP) plot of CD8 T cells isolated from p53-suppressed proliferating (PRO) and p53-reactivated senescent (SEN) tumors. Right, gene set enrichment analysis of T-cell exhaustion marker genes in CD8+ T cells from proliferating (p53-suppressed) versus senescent (p53-reactivated) tumors. NES, normalized enrichment score; Pval, P value. G, UMAP plot of the expression of selected genes (Cd8a, Cd44, Tnfrsf9, Cd69, Tox, and Fasl) between CD8 T cells isolated from senescent (p53-reactivated) and proliferating (p53-suppressed) tumors. H, Representative immunofluorescence images of CD8 T cells and F4/80-positive macrophage staining in the orthotopic NSP liver tumor. Tumor samples were collected 9 days after Dox withdrawal. Data are presented as mean ± SEM. All scale bars, 100 μm. A two-tailed Student t test was used. *, P < 0.05; **, P < 0.01.

Cellular senescence is characterized by stable cell-cycle arrest and a secretory program that modulates the tissue microenvironment1,2. Physiologically, senescence serves as a tumour-suppressive mechanism that prevents the expansion of premalignant cells3,4 and has a beneficial role in wound-healing responses5,6. Pathologically, the aberrant accumulation of senescent cells generates an inflammatory milieu that leads to chronic tissue damage and contributes to diseases such as liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis1,7. Accordingly, eliminating senescent cells from damaged tissues in mice ameliorates the symptoms of these pathologies and even promotes longevity1,2,8-10. Here we test the therapeutic concept that chimeric antigen receptor (CAR) T cells that target senescent cells can be effective senolytic agents. We identify the urokinase-type plasminogen activator receptor (uPAR)11 as a cell-surface protein that is broadly induced during senescence and show that uPAR-specific CAR T cells efficiently ablate senescent cells in vitro and in vivo. CAR T cells that target uPAR extend the survival of mice with lung adenocarcinoma that are treated with a senescence-inducing combination of drugs, and restore tissue homeostasis in mice in which liver fibrosis is induced chemically or by diet. These results establish the therapeutic potential of senolytic CAR T cells for senescence-associated diseases.

Abstract While the majority of human pancreatic cancers are classified as ductal adenocarcinomas, the cell of origin of these tumors remains uncertain and many tumors are comprised of dedifferentiated cell types, making it imperative to understand the full spectrum of neoplastic differentiation and how it is regulated. We have developed a new zebrafish model of pancreatic cancer driven by tissue-specific expression of UAS:mK02-KRASG12D under the regulation of a Ptf1a:Gal4/VP16 transcriptional driver. Transgenic zebrafish generate mixed tumors comprised histologically of mixed acinar and ductal elements, providing the opportunity to interrogate specific transcriptional and epigenetic modifiers responsible for regulating neoplastic pancreatic cellular differentiation. Single-cell RNA sequencing of these tumors has confirmed neoplastic cell populations expressing exclusively acinar and ductal markers, as well as distinct clusters expressing markers of both cell lineages. Screening a panel of 21 unique chromatin modifiers and chromatin readers for differential expression in these distinct cell clusters reveals hdac9b to be upregulated in the progenitor-like ductal population. Gain- and loss-of-function genetic studies will be used to determine the functional role of hdac9b in generating distinct neoplastic cell differentiation states. We are also generating pseudo-time trajectory inferences to further determine the directionality of differentiation states in these cells. By generating new insights into regulation of distinct differentiation states in pancreatic cancer, we hope that new strategies for therapeutic manipulation of neoplastic cell differentiation will emerge. Citation Format: Somer M. Matar, Britton C. Goodale, Xiaoying Liu, Annie Katanga, Nicole F. Gallien, Sandra Blazquez, Direna Alonso-Curbelo, Steven D. Leach. Single-cell profiling of neoplastic cell populations in a KRAS-initiated zebrafish pancreatic cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B112.

IFNγ signaling in senescent tumor cells is necessary for immune surveillance. A,Ifngr1 KO of both proliferating and senescent NSP cells validated by flow cytometry. B, Tumor regression phenotype of Ifngr1 KO or control sgRNA–transfected tumor cells orthotopically injected into Bl/6N mice upon p53 restoration. A control sgRNA targeting a gene desert located on Chr8 (Ctrl KO) serves as a control. C, Tumor regression phenotype of parental NSP tumor cells orthotopically injected into WT or Ifng KO mice upon p53 restoration. D, Representative macroscopic images of tumor collected at day 21 after p53 restoration from C. E, Flow cytometry analysis of CD45 abundance in tumor from indicated groups. F, Representative immunofluorescence in p53-suppressed (proliferating) and p53-restored (senescent, 7 days after p53 restoration) tumor from the indicated host. NSP tumor cells were transduced with GFP-expressing vector for visualization. Scale bars, 50 μm. Data are presented as mean ± SEM. Two-tailed Student t test was used. **, P < 0.01; ***, P < 0.001.

Cancer is a progressive disease that can develop and evolve over decades, with inflammation playing a central role at each of its stages, from tumor initiation to metastasis. In this context, macrophages represent well-established bridges reciprocally linking inflammation and cancer via an array of diverse functions that have spurred efforts to classify them into subtypes. Here, we discuss the intertwines between macrophages, inflammation, and cancer with an emphasis on temporal dynamics of macrophage diversity and functions in pre-malignancy and cancer. By instilling temporal dynamism into the more static classic view of tumor-associated macrophage biology, we propose a new framework to better contextualize their significance in the inflammatory processes that precede and result from the onset of cancer and shape its evolution.

Senescence remodels tissue-sensing programs and cell-surfaceome landscape. A, Gene set enrichment analysis (Reactome) of RNA-seq data from proliferating (PRO, p53 off) versus senescent (SEN, p53 on for 8 days) NSP liver tumor cells in vitro. NES, normalized enrichment score. B, Subcellular localization of DEGs (P < 0.05; fold change > 2) in all detected genes [transcripts per kilobase million (TPM) > 1] from RNA-seq. C, Gene ontology (GO) analysis of DEGs encoding PM proteins upregulated in senescent cells. TM, transmembrane. D, Transcriptomic analysis of all DEGs (proliferating vs. senescent) in the presence or absence of JQ1 treatment. The C1 cluster (in red) contains the senescence-specific genes sensitive to JQ1, and the C4 cluster (in blue) contains the proliferation-specific genes sensitive to JQ1. E, Meta-analysis of RNA-seq dataset from SENESCopedia by performing subcellular localization of DEGs (same as Fig. 2D) and Fisher exact test to examine the relative enrichment of upregulated and downregulated EC/PM-DEGs deviated from the random distribution. See also Supplementary Fig. S7E and S7F. F, Mass spectrometry (MS) analysis of PM-enriched proteome in proliferating and senescent cells. Protein level is normalized to mean expression of the protein of all samples. Controls are the samples without biotin labeling serving as background. Red and blue boxes represent proteins enriched in senescent and proliferating cells, respectively. n = 6 for both the senescent and proliferating experimental groups, and n = 3 and 4, respectively, for their control. G, Distribution of upregulated and downregulated GeneCards-annotated PM proteins profiled by MS. NC, no change. H, Volcano plot of GeneCards-annotated PM proteins profiled by MS.