Understanding the cells of origin is essential for overcoming therapy resistance in esophageal squamous cell carcinoma (ESCC). We utilized machine learning-based single-cell trajectory analysis on 4NQO-induced murine models and genetically engineered organoids to identify multiple distinct cell clusters that serve as cellular origins of ESCC. Gene regulatory network analysis of these populations indicated activation of stem/progenitor cell regulators, including PRRX2 and CEBPβ. Translating these findings, a transcriptome-based drug repurposing screen identified five chemical candidates, four of which are potent Cyclin-Dependent Kinase (CDK) inhibitors, aligning with the frequent loss-of-function mutations in TP53 and CDKN2A observed in ESCC. Notably, CDK inhibitors markedly inhibit ESCC cell proliferation. This research delineates the potential cellular origins of ESCC and their key regulons, thereby pioneering a single-cell-derived therapeutic strategy that exposes vulnerabilities in tumor-initiating cells.

Preclinical cancer models often use subcutaneous (SC) implantation, which fails to recapitulate the native tumor microenvironment (TME) of orthotopic (ORT) sites. To resolve these differences, we used single-cell RNA sequencing (scRNA-seq) on paired SC and ORT implants of the CKP syngeneic gastric cancer model. Histopathological differences were minimal, but scRNA-seq revealed profound TME divergence. ORT tumors displayed robust stromal activation, coordinated fibroblast and endothelial signaling, and an immune compartment marked by higher T/NK cell activation and IgA-biased B cell plasma programs, reflecting a physiological mucosal environment. In contrast, SC tumors had higher overall T cell infiltration but showed markedly increased CD8+ T cell exhaustion and an enriched oxidative tumor program. Our findings provide critical guidance: SC models are optimal for high-throughput and exhaustion-focused assays, whereas ORT models are indispensable for studying organ-specific immune and stromal biology with translational fidelity.

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SMARCA4 and other components of the SWI/SNF chromatin remodeling complex have been implicated in various cancers. Yet, its role in small cell lung cancer (SCLC) tumorigenesis remains poorly understood. Genetically engineered mouse models (GEMMs) of SCLC revealed that deletion of Smarca4 significantly decreased tumor development in this model. Pharmacological inhibition of SMARCA4 decreased the proliferation of preneoplastic neuroendocrine (NE) cells. These effects coincided with reduced expression of the lineage-specific transcription factor, ASCL1, suggesting that disruption of the SMARCA4-ASCL1 axis impairs tumor development. However, Smarca4-deficient tumors, albeit smaller than controls, displayed features associated with malignant progression, including variant histology and the loss of NE differentiation. This prompted us to test the functional role of SMARCA4 in established tumor cells that recapitulate late-stage disease. Intriguingly, whilst Smarca4 knockdown in tumor cells failed to affect their proliferative capacity in vitro, Smarca4 knockdown tumors exhibited enhanced growth following subcutaneous transplantation in athymic nude mice. Interestingly, SMARCA4 knockdown significantly reduced expression and cell-surface display of PVR, a ligand for activating natural killer (NK) cells. These results led to an idea that the enhanced tumor formation was partly owing to altered tumor-NK cell interactions mediated by the SMARCA4-PVR axis in tumor cells. These findings suggest that SMARCA4 plays a temporally distinct role in SCLC, supporting early tumorigenesis but potentially functioning as a tumor suppressor in the later stages. The dramatic differences observed when targeting SMARCA4 in distinct disease states emphasize a need to acknowledge how differences in the timing of alterations can drastically alter tumor evolution.

Background: Mucinous gastric adenocarcinoma (MGC) represents an aggressive subtype associated with a poor prognosis and a limited response to immunotherapy. CRACD, an actin polymerization, is frequently inactivated in gastric cancer (GC). However, CRACD’s role in GC tumorigenesis remains unknown. Objective: To determine the impact of CRACD loss on gastric tumorigenesis and identify potential therapeutic vulnerabilities. Design: Genetically engineered gastric organoids and syngeneic/orthotopic mouse models were employed to investigate the impact of CRACD inactivation. Multi-omics analyses (scRNA-seq and ATAC-seq) alongside pharmacological inhibition were conducted, with subsequent validation using patient-derived organoids and tissue microarrays. Results: CRACD loss induced mucinous cell plasticity, epithelial disruption, and immune evasion. Mechanistically, actin dysregulation activated NF-κB/COX2 signaling and ROS production, stabilizing HIF-1α for PD-L1 transactivation. Pharmacological blockade of HIF-1α or PD-L1 restored immune surveillance and suppressed tumor growth. Clinically, CRACD downregulation correlated with PD-L1 upregulation in GC tissues. Conclusion: CRACD inactivation is sufficient to develop a MGC subtype with immune evasion through the ROS–HIF-1α–PD-L1 axis. CRACD status may serve as a biomarker for targeted (HIF-1α) or immunotherapies (PD-L1).

Although histologically normal, esophageal preneoplastic cells harbor early genetic alterations and likely exhibit lineage plasticity. However, their origins and trajectories remain unclear. To address this, we combined genetic barcoding with single-cell RNA sequencing to trace the lineage of esophageal preneoplastic cells. We identified a distinct progenitor-like cell population with high plasticity. Through a newly developed scoring system, these high-plasticity cells are mapped, revealing their contributions to proliferative and basal cell populations. This approach uncovers molecular markers, including Nfib and Qk, that define these precursor cells, validated by spatial transcriptomics and a Trp53 Cdkn2a Notch1 mouse model. These findings provide critical insights into early tumorigenesis, highlighting the potential of precursor cells as biomarkers for early detection and therapeutic targets of esophageal squamous cell cancer. By elucidating the cellular dynamics underlying esophageal preneoplasia, this research lays the foundation for strategies to prevent malignant progression, offering broader implications for improving cancer diagnostics and treatment approaches.

Small cell lung cancer (SCLC) is aggressive with limited therapeutic options. Despite recent advances in targeted therapies and immunotherapies, therapy resistance is a recurring issue, which might be partly due to tumor cell plasticity, a change in cell fate. Nonetheless, the mechanisms underlying tumor cell plasticity and immune evasion in SCLC remain elusive. CRACD, a capping protein inhibitor that promotes actin polymerization, is frequently inactivated in SCLC. Cracd knockout (KO) transforms preneoplastic cells into SCLC tumor-like cells and promotes in vivo SCLC development driven by Rb1, Trp53, and Rbl2 triple KO. Cracd KO induces neuroendocrine (NE) plasticity and increases tumor cell heterogeneity of SCLC tumor cells via dysregulated NOTCH1 signaling by actin cytoskeleton disruption. CRACD depletion also reduces nuclear actin and induces EZH2-mediated H3K27 methylation. This nuclear event suppresses the MHC-I genes and thereby depletes intratumoral CD8+ T cells for accelerated SCLC tumorigenesis. Pharmacological blockade of EZH2 inhibits CRACD-negative SCLC tumorigenesis by restoring MHC-I expression and immune surveillance. Unsupervised single-cell transcriptomics identifies SCLC patient tumors with concomitant inactivation of CRACD and downregulated MHC-I pathway. This study defines CRACD, an actin regulator, as a tumor suppressor that limits cell plasticity and immune evasion and proposes EZH2 blockade as a viable therapeutic option for CRACD-negative SCLC.

Esophageal organoids serve as powerful systems to study epithelial lineage hierarchies and cancer biology. Gene manipulation in these organoids has traditionally involved overexpression or knockout strategies. However, CRISPR/Cas9-based knock-in (KI) approaches now enable precise cell lineage tracing and live imaging. Here, we describe protocols to generate fluorescent KI organoids from murine esophageal epithelium by tagging Krt13 (BFP) and Sox2 (mNeon). These dual-reporter organoids allow direct monitoring of growth dynamics and differentiation trajectories. We outline CRISPR/Cas9 design, donor construction using homology-independent approaches (CRISPaint), delivery into organoid cells, enrichment and single-clone isolation, and validation by fluorescence. For organoids, homology-directed repair (HDR) can be relatively inefficient to deliver the reporter frame. Thus, we highlight the practical advantages of non-homologous end joining (NHEJ)-based methods, which enable robust, frame-accurate KI with minimal cloning. The methods outlined here can be applied broadly for cell lineage tracing, damage-response studies, and cancer modeling.

Spatiotemporal gene expression is the fundamental feature of cellular differentiation, including neuron differentiation. The epigenetic mechanism underlying spatiotemporal gene regulation during in vivo neuron differentiation remains largely unknown. Granule cells (GCs) constitute the vast majority of neurons in the cerebellum, which contains most of neurons in the brain. Here, we show that Atoh1-Cre‒mediated knockout (ACKO) of Kmt2d encoding the lysine methyltransferase KMT2D (MLL4) in cerebellar GC lineage inhibits the transition of GC progenitors to GCs while cell-non-autonomously impacting other cerebellar cells. Kmt2d ACKO impaired cerebellum-associated behaviors and caused facial peculiarity, microcephaly, and reduced body size in mice. KMT2D temporally activated neuronal differentiation programs in cerebellar GCs. KMT2D-mediated activation of the key neuronal transcription factor genes En2, Pax6, and Myt1l via super-enhancer/enhancer programming was critical for GC differentiation. These findings reveal a unique epigenetic mechanism in which KMT2D temporally orchestrates gene expression required for cerebellar GC differentiation by programming neuronal enhancers.

Background: Chronic pain and cancer interact bidirectionally, with pain enhancing sensory peptides and potentially promoting tumor growth. Despite this, most chemotherapy-induced neuropathic pain (CIPN) studies overlook the contribution of cancer itself to neuropathy, focusing instead on chemotherapy-induced mechanisms. Animal models of chemotherapy-induced neuropathic pain (CINP) have been developed by injecting chemotherapeutic drugs such as paclitaxel into normal animals without cancer. This study aimed to develop a new model in mouse mammary tumor virus–polyomavirus middle T antigen (MMTV-PyMT) mice, a widely used breast cancer model with normal immune function. Results: The percentage of positive response (PPR) of paclitaxel-injected MMTV-PyMT mice increased (about 20%; baseline, 10%) on day 4, reached the highest levels (50%-60%) on days 6-9, and then plateaued by day 29. In comparison, the PPR of paclitaxel-injected C57BL/6 was less than 10% on days 0-6, was about 40% on day 9, and then plateaued by day 29. Breast tumor–bearing mice exhibited an earlier onset and greater severity of paclitaxel-induced pain behaviors than tumor-free C57BL/6 mice. Systemic LGK-974 ameliorated paclitaxel-induced pain behaviors in MMTV-PyMT mice. Active β-catenin was detected in neurons and satellite cells of the dorsal root ganglia. Conclusions: Paclitaxel-induced neuropathic pain model in breast tumor–bearing female MMTV-PyMT mice may be a useful animal model for investigating the analgesic effects and underlying mechanisms for CINP in breast cancer patients as well as the interplay between CINP development and cancer progression.

Organoids are widely used for disease modeling due to their faithful recapitulation of tissue homeostasis, regeneration, and disease processes. While organoids are typically cultured under stemness-promoting conditions with several growth factors and chemicals, these stimulated stem cell niches may not accurately represent the regenerative environment. Herein, we present a detailed, efficient protocol for generating and culturing murine lung organoids (LOs) that mimic regeneration. We also describe how to establish genetically engineered LOs using viral transduction.

Cell plasticity, changes in cell fate, is crucial for tissue regeneration. In the lung, failure of regeneration leads to diseases, including fibrosis. However, the mechanisms governing alveolar cell plasticity during lung repair remain elusive. We previously showed that PCLAF remodels the DREAM complex, shifting the balance from cell quiescence towards cell proliferation. Here, we found that PCLAF expression is specific to proliferating lung progenitor cells, along with the DREAM target genes transactivated by lung injury. Genetic ablation of Pclaf impaired AT1 cell repopulation from AT2 cells, leading to lung fibrosis. Mechanistically, the PCLAF-DREAM complex transactivates CLIC4, triggering TGF-beta signaling activation, which promotes AT1 cell generation from AT2 cells. Furthermore, phenelzine that mimics the PCLAF-DREAM transcriptional signature increases AT2 cell plasticity, preventing lung fibrosis in organoids and mice. Our study reveals the unexpected role of the PCLAF-DREAM axis in promoting alveolar cell plasticity, beyond cell proliferation control, proposing a potential therapeutic avenue for lung fibrosis prevention.

Understanding the mechanisms underlying immune evasion is crucial for developing novel anticancer modalities. To systematically uncover tumor-intrinsic genetic modulators involved in immune escape in tumor microenvironment, we performed genome-scale in vivo CRISPR screens in two syngeneic models and later expanded up to seven syngeneic models with a focused validation library. These data help us better understand tumor immune evasion and pave the way for developing effective therapeutics. Importantly, we uncovered that Mga depletion elicited an antitumor immune response and inhibited tumor growth in triple-negative breast cancer. Our findings suggest that Mga may play a role in modulating the tumor immune landscape, though the precise mechanisms require further investigation. Further studies are needed to test MGA inhibition in cancer

Tumor cell plasticity contributes to intratumoral heterogeneity and therapy resistance. Through cell plasticity, some lung adenocarcinoma (LUAD) cells transform into neuroendocrine (NE) tumor cells. However, the mechanisms of NE cell plasticity remain unclear. CRACD, a capping protein inhibitor, is frequently inactivated in cancers. CRACD knock-out (KO) is sufficient to de-repress NE-related gene expression in the pulmonary epithelium and LUAD cells. In LUAD mouse models, Cracd KO increases intratumoral heterogeneity with NE gene expression. Single-cell transcriptomic analysis showed that Cracd KO-induced NE cell plasticity is associated with cell de-differentiation and stemness-related pathway activation. The single-cell transcriptomic analysis of LUAD patient tumors recapitulates that the distinct LUAD NE cell cluster expressing NE genes is co-enriched with impaired actin remodeling. This study reveals the crucial role of CRACD in restricting NE cell plasticity that induces cell de-differentiation, providing new insights into the cell plasticity of LUAD.

Despite the promising outcome of immune checkpoint blockade (ICB), ICB resistance is a new challenge. Thus, selecting patients for specific ICB applications is crucial for maximizing therapeutic efficacy. Herein we curated 69 human esophageal squamous cell cancer (ESCC) patients’ tumor microenvironment (TME) single-cell transcriptomic datasets for ESCC subtyping. Notably, integrative analyses of the cellular network transcriptional signatures of T cells, myeloid cells, and fibroblasts define distinct ESCC subtypes characterized by T cell exhaustion, Interferon alpha and beta signaling, TIGIT enrichment, and specific marker genes. Furthermore, this approach classifies ESCC patients into ICB responders and non-responders, validated by liquid biopsy single-cell transcriptomics. This study stratifies ESCC patients by TME transcriptional network, which provides a novel insight into tumor niche remodeling and helps predict ICB responses of ESCC patients.

Diffuse-type gastric adenocarcinoma (DGAC) is a deadly cancer often diagnosed late and resistant to treatment. While hereditary DGAC is linked to CDH1 mutations, the role of CDH1/E-cadherin inactivation in sporadic DGAC tumorigenesis remains elusive. We discovered CDH1 inactivation in a subset of DGAC patient tumors. Analyzing single-cell transcriptomes in malignant ascites, we identified two DGAC subtypes: DGAC1 (CDH1 loss) and DGAC2 (lacking immune response). DGAC1 displayed distinct molecular signatures, activated DGAC-related pathways, and an abundance of exhausted T cells in ascites. Genetically engineered murine gastric organoids showed that Cdh1 knock-out (KO), KrasG12D, Trp53 KO (EKP) accelerates tumorigenesis with immune evasion compared to KrasG12D, Trp53 KO (KP). We also identified EZH2 as a key mediator promoting CDH1 loss-associated DGAC tumorigenesis. These findings highlight DGAC's molecular diversity and potential for personalized treatment in CDH1-inactivated patients.

Background and aims: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown. Methods: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids (EOs). Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing (scRNA-seq). Tumorigenicity of organoids and tumor-infiltrated immune cells were monitored by allograft transplantation. Human ESCC scRNA-seq datasets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion. Results: We established 32 genetically engineered EOs and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple knockout (KO) induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN KO also generates immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via NF-B. Moreover, comparative single-cell transcriptomic analyses stratified ESCC patients and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. Conclusions: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable approach to target PCN-type ESCC.