Abstract LB-146: Identification of the tumor transition states occurring during EMT and their implications for metastasis

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Identifying transitional states is crucial for understanding protein conformational changes that underlie numerous biological processes. Markov state models (MSMs), built from Molecular Dynamics (MD) simulations, capture these dynamics through transitions among metastable conformational states, and have demonstrated success in studying protein conformational changes. However, MSMs face challenges in identifying transition states, as they partition MD conformations into discrete metastable states (or free energy minima), lacking description of transition states located at the free energy barriers. Here, we introduce Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR), a deep learning framework inspired by out-of-distribution (OOD) detection in trustworthy artificial intelligence (AI). TS-DAR offers an end-to-end pipeline that can simultaneously detect all transition states between multiple free minima from MD simulations using the regularized hyperspherical embeddings in latent space. The key insight of TS-DAR lies in treating transition state structures as OOD data, recognizing that they are sparsely populated and exhibit a distributional shift from metastable states. We demonstrate the power of TS-DAR by applying it to a 2D potential, alanine dipeptide, and the translocation of a DNA motor protein on DNA, where it outperforms previous methods in identifying transition states.

States of Transition takes a deep dive into the multiple roles states are playing in supporting transitions to a more sustainable world and where there is scope for their transformation. Going beyond unhelpful binaries - which cast the state as the central problem or the all-encompassing solution to ecological and social crises - it explores diverse current state practice across key domains: military, democracy, welfare, entrepreneurial, industrial, and foreign policy. It builds on theoretical resources from a range of disciplines, as befits the challenge of making sense of these diverse aspects of state power. It moves beyond existing analysis of the 'environmental state' to explore scope for a 'transition state' to emerge, capable of corralling and transforming all aspects of state power behind the goal of responding to the existential threat of planetary collapse. The book will be invaluable to students, academics, and practitioners concerned with environmental policy and sustainability.

Does (H5O2)+− X (X = Ar, Ne, He) complex with C2 symmetry really exist?

BackgroundBladder cancer (BC) is a prevalent malignancy characterized by significant cellular heterogeneity. While single-cell multi-omics studies have provided valuable insights, much of the existing data remains underexplored, limiting our understanding of BC’s molecular mechanisms. Uncovering the pathogenesis of BC and finding new treatment methods are urgent problems to be solved. This study aims to address this gap by re-analyzing available single-cell datasets to uncover novel insights into BC.MethodsIn this study, we retrieved three single-cell transcriptome datasets by searching the Gene Expression Omnibus (GEO) database, focusing on single-cell sequencing of normal mouse bladder within the past 5 years. Through quality control and batch effect elimination, we obtained a total of 24,930 cells including epithelial, stromal, and immune cells. Subgroup analysis, pseudotemporal analysis, cell–cell communication, and transcription factor analysis were conducted specifically on epithelial cells to identify a transitional state during basal cell differentiation. We further compared the expression profiles of key transcription factors in cancer and normal tissues. In addition, we also performed immunohistochemical staining and survival analysis for key transcription factors.ResultsSubgroup analysis revealed multiple subtypes of epithelial cells, including basal, umbrella, and intermediate cells. Through pseudotemporal analysis, we discovered the developmental trajectory from basal cells to umbrella cells and further found that Basal_I is a transitional state for basal cell differentiation. Cell-to-cell communication analyses highlighted the pivotal role of Basal_I in cell–cell interactions, and key ligand-receptor pairs associated with cancer progression were also identified. Furthermore, elevated expression levels of key transcription factors in Basal_I were found to be closely associated with the stage and prognosis of BC. Immunohistochemical staining results further confirmed the upregulated expression of these transcription factors in BC.ConclusionsCollectively, we found a transitional state of basal cells in normal bladder epithelial cells in mice, which may be related to the occurrence and development of BC, providing important clues for further understanding of the pathogenesis of BC. Our study provided possible molecular mechanisms or target for the research and treatment of BC.

Elucidating the Characteristics of Two Tumor Cell Populations in Small Cell Variant of Anaplastic Lymphoma Kinase-Positive Anaplastic Large Cell Lymphoma

Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.

Metastatic spread is a complex process based on manyfold interactions of the tumor cells with each other and with the surrounding stroma. In experimental systems tumor cell heterogeneity and presence of various subpopulations which interact with one another to stabilize their relative proportions within the population has been shown. Here we report a patient with melanoma, showing morphological evidence of two distinct tumor cell populations in the primary tumor and in all subcutaneous metastases. By image analysis, both populations were clearly characterized by minimal nuclear diameter and by nuclear form factor and were demonstrable in each specimen. The fact that these peculiar cell populations were present in all melanoma lesions removed from the patient might indicate that the populations require the presence of each other and that none of them is metastatically competent on its own.

Intratumoral heterogeneity in pancreatic ductal adenocarcinoma (PDAC) is characterized by the coexistence of classical epithelial, basal, and mesenchymal cancer cell states. Targeting cancer cell plasticity—the ability of tumor cells to adapt to extrinsic pressures via non-genetic mechanisms—has emerged as a promising therapeutic strategy. In parallel, the immunosuppressive tumor microenvironment remains a major barrier to effective treatment in PDAC. Yet, whether specific cell states possess distinct functional properties that drive key tumor behaviors—such as phenotypic plasticity or immune evasion—remains poorly understood. Using genetically engineered mouse models driven by oncogenic Kras G12D and loss of Trp53 (“KPC” model), we profiled primary PDAC tumors using single-cell RNA sequencing and single cell ATAC sequencing. We find that the KPC model harbors a basal state, which harbors transcriptional signature that aligns with the "basal" signature in human PDAC and is linked to poor prognosis and chemoresistance. The basal state co-expresses both epithelial and mesenchymal programs and has a distinct epigenetic landscape with accessible chromatin across gene sets that mark cancer cell states with divergent phenotypes, suggesting that this cell state is primed for cell state switching. To functionally interrogate the basal state in PDAC progression, we developed a novel lineage-tracing and ablation system. Lineage-tracing experiments revealed that the basal state has a robust capacity for differentiation into epithelial and mesenchymal states, indicating that the basal state is functionally plastic and acts as a source of fixed malignant states. Strikingly, targeted elimination of the basal state over short time periods resulted in tumor collapse, underscoring a critical role in tumor maintenance. Further, we find that the basal state is a key driver of therapeutic resistance: rather than being eliminated by treatment, it persists through phenotypic adaptation in response to both KRAS-targeted therapies and cytotoxic chemotherapy. Co-elimination of the basal state alongside these therapies leads to deeper and more durable responses, underscoring its functional role in tumor persistence under therapy. In parallel, we observe that the basal state plays a central role in enforcing immunosuppression in PDAC tumors. Basal state ablation rapidly reshapes the tumor immune microenvironment, and we observe loss of cancer-associated fibroblasts, polarization of PDAC-resident macrophages toward a pro-inflammatory phenotype, and increased infiltration of T and NK cells. Cytokine profiling of basal-ablated tumors revealed a dramatic reduction in multiple immune-modulatory and immunosuppressive cytokines. Taken together, our data indicate that PDAC harbors a striking dependency on the basal state for both therapeutic resistance and immune evasion, and encourages therapeutic approaches aimed at eradicating this critical subset of cancer cells. Citation Format: Anupriya Singhal, Kate Ryan, Samuel Rose, Hannah C. Styers, Nikhita Pasnuri, Jonathan Rub, Jung Yun Kim, Ashlyn Moore, Jill Hallin, Zeynep C. Tarcan, Olca Basturk, Rona Yaeger, James G. Christensen, Yan Yan, Elisa de Stanchina, Dana Pe'er, Tuomas Tammela. Functional Dissection of the Highly Plastic Basal Cell State in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B108.

It is generally accepted that primary tumors become heterogeneous as a consequence of tumor-cell genetic instability. Clonal dominance has been shown to occur in some experimental models allowing a subpopulation of cells to overgrow the primary heterogeneous tumor and to metastasize. Alternatively, interactions among coexisting tumor subpopulations may contribute to the emergence of a malignant invasive primary solid tumor. We asked the question whether emergence of carcinoma cells producing a growth/dissociating factor within a tumor cell population may be a determinant for tumor progression and for clonal dominance. To mimic such a situation, we have investigated the impact of tumor subpopulation heterogeneity in an in vivo model in which mixtures of carcinoma cells that differ in their ability to produce acidic fibroblast growth factor are injected into nude mice. Our data indicate that a growth-factor-producing cell subpopulation can confer increased tumorigenicity to an entire cell population and subsequently elicit a shorter delay for appearance of metastasis. A community effect via cell interactions may account for a heterogeneous tumor cell population rather than clonal dominance during progression of certain tumor types.

Specific populations of highly tumorigenic cells are thought to exist in many human tumors, including pancreatic adenocarcinoma. However, the clinical significance of these tumor-initiating (ie, cancer stem) cells remains unclear. Aldehyde dehydrogenase (ALDH) activity can identify tumor-initiating cells and normal stem cells from several human tissues. We examined the prognostic significance and functional features of ALDH expression in pancreatic adenocarcinoma. ALDH expression was analyzed by immunohistochemistry in 269 primary surgical specimens of pancreatic adenocarcinoma and examined for association with clinical outcomes and in paired primary tumors and metastatic lesions from eight pancreatic cancer patients who had participated in a rapid autopsy program. The clonogenic growth potential of ALDH-positive pancreatic adenocarcinoma cells was assessed in vitro by a colony formation assay and by tumor growth in immunodeficient mice (10-14 mice per group). Mesenchymal features of ALDH-positive pancreatic tumor cells were examined by using quantitative reverse transcription-polymerase chain reaction and an in vitro cell invasion assay. Gene expression levels and the invasive potential of ADLH-positive pancreatic cancer cells relative to the bulk cell population were examined by reverse transcription-polymerase chain reaction and an in vitro invasion assays, respectively. All statistical tests were two-sided. ALDH-positive tumor cells were detected in 90 of the 269 primary surgical specimens, and their presence was associated with worse survival (median survival for patients with ALDH-positive vs ALDH-negative tumors: 14 vs 18 months, hazard ratio of death = 1.28, 95% confidence interval = 1.02 to 1.68, P = .05). Six (75%) of the eight patients with matched primary and metastatic tumor samples had ALDH-negative primary tumors, and in four (67%) of these six patients, the matched metastatic lesions (located in liver and lung) contained ALDH-positive cells. ALDH-positive cells were approximately five- to 11-fold more clonogenic in vitro and in vivo compared with unsorted or ALHD-negative cells, expressed genes consistent with a mesenchymal state, and had in vitro migratory and invasive potentials that were threefold greater than those of unsorted cells. ALDH expression marks pancreatic cancer cells that have stem cell and mesenchymal features. The enhanced clonogenic growth and migratory properties of ALDH-positive pancreatic cancer cells suggest that they play a key role in the development of metastatic disease that negatively affects the overall survival of patients with pancreatic adenocarcinoma.

Pancreatic cancer is a devastating disease that usually presents at a late stage that is not amenable to curative treatments. It is notoriously resistant to chemotherapy and radiation, and it is clear that new treatment strategies are needed to positively impact the dismal outcomes associated with this disease. While the initial dogma of tumor biology viewed all tumor cells as equal, recent findings in the field have lead to the discovery of heterogeneous populations of tumor cells, first in blood-borne derived cancers and now within multiple solid tumor organ systems that display a differential ability to repopulate the original tumor. Conceptualized from this finding was the stem-cell hypothesis for cancer, which suggests that only a specific subset of cancer cells within each tumor is responsible for tumor initiation and propagation, termed tumor initiating cells or cancer stem cells (CSCs). By using specific cell surface marker analysis, we have identified distinct populations of tumor cells from primary pancreatic adenocarcinomas that express the surface markers CD44, CD24, and ESA, which are responsible for the initiation of new tumor growth and exhibit characteristics that support the CSC hypothesis. The identification of these cells has opened up an exciting new paradigm on how we might view and devise new patient treatments for this devastating disease. Current investigations of these pancreatic CSCs have identified such signaling pathways as the Hedgehog and BMI-1 signaling pathways which are aberrantly regulated in this select population of cells. Further study of these CSCs and the important signaling pathways that control their function will provide us with powerful information on targeting new therapies to improve patient outcomes.

Binding of Factor VIII to D'D3 and A2 Domains of Von Willebrand Factor Facilitates Mechanical Unraveling of the A2 Domain

The ring-closing reaction of chloride polyether polyol was calculated by Gaussian03 software. The Density Function Theory (DFT) method were employed to study the geometry structures of chloride polyether polyol and the product was obtained on the base of B3LYP/6-31G+ model in this paper. The transitional states (Ts1, Ts2) during the ring-closing process were found by TS method and the energy changing of the system was proved by IRC calculation. Results showed that the ring-closing of chloride polyether polyol had two transition states: hydrogen transferring and the formation of new carbon-oxygen bond. The total energy of reactants was -1459.99627a.u and product is -1460.31170 a.u. The energy of the system was reduced by 175.47621kJ/mol. The ring-closing of chloride polyether polyol was exothermic and stable in thermodynamics.

The three distinct medicinal parts of Angelica sinensis (Oliv.) Diels (Ang) roots are the head, body, and tail (ARH, ARB, and ART, respectively). How endophytic fungi shape the differences in metabolic components among these parts remains unclear. We quantified the distribution of active components and endophytic fungi along the ARH, ARB, and ART and their relationships. Based on the metabolic components and their abundances detected via non-target metabolism, the different medicinal parts were distinguishable. The largest number of dominant metabolic components was present in ART. The difference between ART and ARH was the greatest, and ARB was in a transitional state. The dominant active molecules in ART highlight their effects in haemodynamics improvement, antibacterial, anti-inflammatory, and hormone regulation, while ARH and ARB indicated more haemostasis, blood enrichment, neuromodulation, neuroprotection and tranquilisation, hepatoprotection, and antitumour activities than that of ART. The ARHs, ARBs, and ARTs can also be distinguished from each other based on the endophytic fungi at the microbiome level. The most dominant endophytic fungi were distributed in ART; the differences between ART and ARH were the largest, and ARB was in a transition state, which is consistent with the metabolite distributions. Structural equation modelling showed that the endophytic fungi were highly indicative of the metabolic components. Correlation analysis further identified the endophytic fungi significantly positively correlated with important active components, including Condenascus tortuosus, Sodiomyces alcalophilus, and Pleotrichocladium opacum. The bidirectional multivariate interactions between endophytic fungi and the metabolic components shape their spatial variations along the longitudinal direction in the Ang root.