Differentiation Status Of Cells Research Articles

DNA Framework-Based Programmable Atom-Like Nanoparticles for Non-Coding RNA Recognition and Differentiation of Cancer Cells.

The cooperative diagnosis of non-coding RNAs (ncRNAs) can accurately reflect the state of cell differentiation and classification, laying the foundation of precision medicine. However, there are still challenges in simultaneous analyses of multiple ncRNAs and the integration of biomarker data for cell typing. In this study, DNA framework-based programmable atom-like nanoparticles (PANs) are designed to develop molecular classifiers for intra-cellular imaging of multiple ncRNAs associated with cell differentiation. The PANs-based molecular classifier facilitates signal amplification through the catalytic hairpin assembly. The interaction between PAN reporters and ncRNAs enables high-fidelity conversion of ncRNAs expression level into binding events, and the assessment of in situ ncRNAs levels via measurement of the fluorescent signal changes of PAN reporters. Compared to non-amplified methods, the detection limits of PANs are reduced by four orders of magnitude. Using human gastric cancer cell lines as a model system, the PANs-based molecular classifier demonstrates its capacity to measure multiple ncRNAs in living cells and assesses the degree of cell differentiation. This approach can serve as a universal strategy for the classification of cancer cells during malignant transformation and tumor progression.

Abstract 4468: Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer

Abstract Hepatocellular carcinoma (HCC) represents a major global health challenge, characterized by its heterogeneity and aggressive nature. Solid evidence has now suggested that tumor growth is driven by subpopulations of tumor cells with less differentiated cell states, called cancer stem cells (CSCs), which govern therapeutic resistance and tumor recurrence. Therefore, characterization of CSCs and their associated oncogenic signaling pathways is crucial to the development of more specific and sensitive HCC prognostic markers and to the design of novel targeted therapies against CSCs for HCC treatment. In search of the altered genes and pathways that are tightly associated with cancer stemness trait in HCC, we exploited the stemness and oncogenic dedifferentiation signature to mine the HCC dataset of the Cancer Genome Atlas. Phosphoinositide signaling was identified to be enriched and downregulated in the transcriptomes of HCC patients with strong stemness and dedifferentiated signature. Subsequent clinical correlation analysis found inositol polyphosphate-5-phosphatase B (INPP5B) to be the top downregulated gene in phosphoinositide signaling with its expression correlated with poor survival in HCC patients. Functional studies showed that INPP5B overexpression reversed the oncogenic and stemness properties while INPP5B knockout promoted tumor growth in immunocompetent mice. Subsequent RNA sequencing and molecular assays revealed INPP5B deregulation to be associated with lipid metabolism remodeling. Collectively, our results suggest an intricate relationship between phosphoinositide signaling and lipid metabolism through the regulation of INPP5B in HCC. The findings may open new avenue for future research aimed at devising targeted therapies to interfere with liver CSCs and enhance the prognosis of HCC patients. Citation Format: Linglin Liu, Yifei Wang, Yunong Xie, Man Tong. Elucidating the phosphoinositide-modulatory function of INPP5B in regulating cancer stemness and tumorigenesis in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4468.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2852.

Abstract 3055: Characterizing the role of HNF4α isoforms in human PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly and recalcitrant malignancy with a 5-year survival rate of 12%. Transcriptomic studies have classified PDAC into a classical or basal-like subtype. The classical subtype has a better prognosis, response to chemotherapy, and express higher levels of endodermal lineage specifiers compared to the basal-like subtype. However, the field lacks a comprehensive understanding of the key subtype-specific regulators. Functional analysis of transcription factors associated with each subtype are needed to elucidate their roles in regulation of PDAC subtypes and to identify therapeutically targetable vulnerabilities. Previous work in our lab demonstrated that HNF4α is a key regulator of the classical subtype. Using genetically engineered mouse models, our lab showed that loss of HNF4α leads to an increase in tumor burden, a shift from glandular to poorly differentiated histology, and a decrease in the classical subtype score. However, while murine PDAC only express one set of HNF4α isoforms, human PDAC express both sets of HNF4α isoforms, suggesting murine PDAC may not fully recapitulate human disease. There are 2 subsets of HNF4α isoforms driven by expression from 2 distinct promoters, P1 and P2. P1 and P2 isoforms are differentially expressed within the gastrointestinal (GI) tract and play dichotomous roles in GI malignancies. In HNF4α-positive human PDAC, P2 isoforms are always expressed but there is variable expression of P1 isoforms. Based on the differential role of HNF4α isoforms in both normal tissue and cancer, we hypothesize that P1 isoforms will restrain tumor growth and promote greater classical/GI differentiation compared to P2 isoforms. We utilized CRISPRi to selectively inhibit transcription at the P1 or P2 promoter in HNF4α-positive, human cell lines expressing both isoforms. To test for sufficiency, we exogenously expressed P1 or P2 isoforms in HNF4α-negative, human cell lines. We then performed Incucyte, CRISPRi competition, and anchorage-independent growth assays. We performed RNA-seq and ChIP-seq on both exogenous and endogenous HNF4α isoforms. Functional assays revealed that HNF4α isoforms regulated growth in a context dependent manner. Exogenous P1 isoforms significantly inhibited growth while loss of endogenous P1 isoforms was dispensable for growth. Exogenous P2 isoforms inhibited growth to a lesser degree while loss of endogenous P2 isoforms led to a significant reduction in growth. Despite ChIP-seq analysis revealing very similar binding profiles, RNAseq analysis revealed that P1 isoforms are significantly more transcriptionally active than P2 isoforms and are stronger inducers of GI differentiation. Taken together, our data demonstrates that P2 isoforms are more compatible with growth in PDAC compared to P1 isoforms, which promote a more differentiated cell state. Mechanistically, this may be due to the increased transcriptional activity of P1 isoforms. Citation Format: Pengshu Fang, Acramul Kabir, Gabriela Fort, Eric L. Snyder. Characterizing the role of HNF4α isoforms in human PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3055.

Abstract 3617: Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients

Abstract Background: Chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) can induce >70% response rates in patients with relapsed/refractory multiple myeloma (RRMM). However, most patients ultimately relapse. There are two FDA-approved CAR T products: ciltacabtagene autoleucel (cilta-cel) and idecabtagene vicleucel (ide-cel), the former having much more durable responses. The mechanisms that mediate more durable responses with these CAR T cells is unknown. Previous studies have shown that specific cell states in the apheresis and infusion products are associated with long-term efficacy of anti-BCMA CAR therapy in MM and the relationship between durability of response and CAR T cell persistence remains unclear. Methods: We analyzed serial bone marrow and PBMC samples from 20 MM patients who received BCMA CAR T therapy, including cilta-cel, ide-cel, and JCARH125. Flow cytometry and single-cell RNAseq were used to evaluate the features of CAR T cells that are associated with durable clinical responses. Results: While CAR T cells were detectable but variable among patients at 1-month post infusion, the frequency of CAR T cells in bone marrow at 1-month post-infusion is positively correlated with progression free survival (PFS) in all MM patients (p=0.009). This association was also evident in patients just treated with cilta-cel (p= 0.008). By 6 months, CAR T cells could still be detected by high-throughput flowcytometry in the cilta-cel-treated patients, but CAR T cells were not detectable in most of the patients receiving the other 2 products. Cilta-cel CAR T cells predominantly possessed an effector-like surface phenotype at 1-moth post infusion in both CD8+ and CD4+ compartments in bone marrow. These CAR T cells shifted to effector memory and central memory T cell states by 6 months, respectively. For ide-cel and JCARH125, patients with more durable responses (>6mos) there was a significantly higher proportion of CAR T cells with CD8+ effector memory-like state at 1-month post infusion. Conclusions: Our data demonstrate that distinct anti-BCMA CAR T cell treatments lead to very different cellular outcomes: Cilta-cel CAR T cell treatment leads to greater expansion and persistence compared to other anti-BCMA CAR T cells. Cilta-cel CAR T cells also shift from the initial effector state to a memory phenotype. These results provide insights into features of more efficacious anti-BCMA CAR T cells that can help guide the future development of more durable treatments for MM. Citation Format: Kai Wu, Karen Law, Guy Ledergor, Chang Liu, Zenghua Fan, Vibha Gurunathan, Averey Lea, Matthew Clark, Serena Kwek, Alexander Cheung, Jeffrey Wolf, Ajai Chari, Anupuma Kumar, Justin Eyquem, Thomas Martin, Lawrence Fong. Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3617.

Abstract 6248: Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis

Abstract Glioblastoma, IDH-wild type (GBM) is the most common malignant and treatment-refractory brain tumor with a dismal prognosis. Despite the identification of various driver genes in previous large-scale genomic analyses, there has been limited progress in the development of novel treatments. Acquiring an extensive understanding of the molecular mechanisms underlying GBM pathogenesis is crucial to improve prognostic outcomes. We analyzed 289 whole-genome sequencing data (WGS) including 159 unpublished deep WGS (≥ ×120 coverage) along with RNA-seq, DNA methylation array, and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to uncover the molecular mechanisms regulating development and progression of GBM. Our deep WGS enables us to delineate a fine view of clonal architecture, where mutational signature varies between clonal and subclonal mutations, suggesting that different mutational processes contribute to GBM pathogenesis depending on its developmental stage. The differentiation status of tumor cells detected by transcriptional deconvolution analysis is associated with multi-layer profiles including genomic alterations, expression subtypes, DNA methylation, and open chromatin status. Tumors predominantly comprised of differentiated cells display genetic and epigenetic profiles that align with the classical subtype, whereas tumors predominantly composed of stem-like cells exhibit profiles consistent with the proneural subtype. ATAC-seq unveils genome-wide chromatin accessibility features associated with gene expression subtypes. Motif enrichment analysis of differentially accessible sites among the subtypes identified specific transcription factors. The proneural subtype is enriched for the SOX10 binding motif, which is associated with regulating cell states, and exhibits higher expression of SOX10 compared to other subtypes. Conversely, the mesenchymal and classical subtypes are enriched for the CREB1 binding motif, which promotes cell proliferation and angiogenesis through TGFβ2 upregulation, wherein TGFβ2 expression is significantly elevated. These findings support a model in which the difference in chromatin structure also regulates the progression of GBM.Our analysis reveals the multi-layer molecular interactions underlying the progression of GBM enhancing our understanding of the pathogenesis. Citation Format: Takuma Nakashima, Yusuke Funakoshi, Ryo Yamamoto, Yuriko Sugihara, Shohei Nambu, Yoshiki Arakawa, Shota Tanaka, Joji Ishida, Ryuta Saito, Ryosuke Hanaya, Koji Yoshimoto, Yoshitaka Narita, Hiromichi Suzuki. Multilayered molecular interactions underlying glioblastoma pathogenesis through multi-omics analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6248.

Abstract 5342: Evaluation of autologous and allogeneic T cell-based therapies in ex-vivo and in-vivo xenograft tumor models

Abstract The field of adoptive T cell-based immunotherapy, including tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR) & Chimeric antigen receptor (CAR) - T cells, has undergone unprecedented growth, specifically later in delivering effective and durable clinical response. Notable success with CAR-T cells is limited to hematological malignancies while clinical gains with solid tumors is still a challenge. Solid tumors’ heterogenous target expression and loss, T cell status and metabolic fitness, T cell trafficking into the tumor, and the hostile immunosuppressive tumor microenvironment (TME) have hampered T cell efficacy in the patients. There is a lack of clinically relevant solid tumor models for the translation of T cell-based therapies into the clinic. We report the development of translational ex-vivo and in-vivo platforms for evaluating cell line-derived and patient-derived xenografts (CDX and PDX) models (BarneyOI™️ model), validated with hCD3 T-cells, autologous TILs and/or allogeneic 3rd generation CAR-T cells (scFv(HER2)-CD28-4-1BB-CD3zeta CAR-T). Anti-hPDL1 but not anti-hPD1 significantly enhanced hCD3 T-cell and autologous TIL-mediated killing of tumor cells in an ex-vivo co-culture assay using Incucyte-based live cell imaging. Also, a dose-dependent killing of HER-2 expressing CDX (A549 & Colo205) and PDX was observed with HER-2 targeting CAR-T cells in an ex-vivo co-culture assay. This was in concordance with the activation, effector function, and differentiation status of the CAR-T cells, immune phenotype by Cytek aurora spectral flow cytometry. Further, to imitate the patient population and the effect of solid TME, HER-2 targeting CAR-T cells were assessed for their in-vivo efficacy and immune response differences in the subcutaneous and orthotopically implanted HER-2 expressing PDX in immunodeficient NOG mice. Our findings highlight the importance of testing T cell-based immune therapies in the appropriate translational tumor models with better certainty of clinical outcome. Citation Format: Sherry Liu, Jasmine Borroel, Carmen Sunico, Emily Eastwood, Bridget Corcoran, Bianca Carapia, Jantzen Sperry, Jonathan Nakashima, Rajeev Shrimali. Evaluation of autologous and allogeneic T cell-based therapies in ex-vivo and in-vivo xenograft tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5342.

Abstract 903: AURIGIN: A comprehensive single-cell OMICs atlas of human development and an AI/ML framework to classify and identify the drivers of tumor plasticity and altered cellular state

Abstract Altered cellular state and plasticity have been increasingly identified as drivers of poor prognosis and treatment-resistance in cancer. Identifying therapeutics that correct altered cellular state and plasticity meets a broad need in the field of oncology, particularly in tumors resistant to standard-of-care therapy. Tumor plasticity and altered cellular states encompass many potential configurations, including dedifferentiated and transdifferentiated tumors, so classifying tumors based on their specific cell state profile is critical to identifying the gene drivers that are specific to that altered state. For that reason, we have developed AURIGIN - a platform that combines a comprehensive single-cell OMICs atlas of human development with an AI/ML framework that can classify cell state plasticity and identify the genes and pathways that drive those specific altered cellular states. AURIGIN is more comprehensive than previously disclosed single cell atlases in that it focuses and enriches the diverse stem, progenitor, and developmental states commonly involved in plastic cell states of cancer. The platform standardizes and integrates these states with multiple atlases of differentiated adult tissues to create a unified atlas of human development. In addition, the AI/ML paradigm of AURIGIN is embedded with developmental biology models that enable it to deconvolute state heterogeneity within tumor indications for precise identification of the most relevant gene targets for novel therapies. AURIGIN also unifies classified plasticity across tissue types by identifying altered cellular states that span multiple indications. Furthermore, we present the implementation of AURIGINDRIVE ML models that integrate pathway information, physical interaction datasets, and regulatory annotations to accurately predict the gene targets at the top of hierarchy of control of tumor plasticity. AURIGIN also enables and accelerates target validation by identifying models that map to the relevant classified plastic states. Similarly, AURIGIN defines clinically relevant patient selection and treatment efficacy biomarkers that are specifically defined from the classified plastic cellular state. We demonstrate the success of AURIGIN for therapeutic discovery. In sum, AURIGIN amplifies the value of multiOMIC tumor data in cellular state and plasticity target discovery by mapping the data into a developmental biology-informed ML framework. Citation Format: Joseph DeBartolo, Henry Wilson, Kimberly S. Straley, Maulasri Bhatta, Sambad Sharma, Sara Sinicropi-Yao, James Neef, Betty Chan, Andrew McRiner, Mark Bittinger, Laura Antipov, Katharine E. Yen, Thomas G. Graeber, David S. Millan. AURIGIN: A comprehensive single-cell OMICs atlas of human development and an AI/ML framework to classify and identify the drivers of tumor plasticity and altered cellular state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 903.

Abstract 1598: Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models

Abstract Background & Aim: Progression and therapeutic resistance in pancreatic cancer depends on the ability of cancer cells to adopt specific cell states. These phenotypes largely depend on environmental tissue niches. Here, we investigated therapy-induced tumor dynamics in vivo at single-cell spatial and temporal resolution in different subtypes of PDAC using several genetically engineered mouse models, which reflect distinct human PDAC ecosystems. Methods & Results: We established orthotopic tumor models of pancreatic cancer with distinct tumor microenvironment (TME) formation (reactive vs deserted stroma), which were monitored for tumor growth to define tumor response patterns under MAPK inhibition. Serial ultrasound-guided biopsy sampling was performed to collect tissues from the same tumors at pre-treatment, responsive and recurrence periods.Transcriptomic analysis of biopsies using the Mfuzz algorithm delineated highly dynamic gene and pathway activities during treatment. Reactive, but not deserted, TME showed an upregulation of immune-related pathways (antigen presentation, T cell receptor, NK cytotoxicity) immediately after MAPK inhibition in regressed tumors that dropped dramatically during recurrence of tumors, in which upregulation of TGF-b signaling was observed. In the deserted stroma model, on the other hand, an enrichment in drug metabolic pathways during recurrence was observed.CAF cluster deconvolution analysis revealed an increase in a LRRC15+ CAF cluster, which was TGF-b-driven and immunosuppressive, during recurrence period only in the reactive model. The dynamic changes of immune cells and CAFs were validated at protein level by multiplex immunofluorescent imaging. Cmputational spatial analysis revealed a tumor-encapsulating and CD8+ cytotoxic T cell-repelling pattern exhibited by LRRC15+FAP+ CAFs, implying a potential role of this CAF subset in T cell suppression during recurrence in reactive subtype. Using spectral cell sorting with CellView image technology, specific CAF subsets, tumor cells as well as immune cell subsets based on 30+ markers and cell morphology were isolated, characterized and assessed by ex vivo functional assays to confirm the immunosuppressive and tumor protective mechanism mediated by CAFs. Conclusion: Longitudinal in vivo assessment of distinct PDAC subtypes provides deep mechanistic insights to accurately unlock specific response patterns of tumors with heterogeneous TME dynamics upon therapeutic perturbation, leveraging identification of key nodes for intervention. Citation Format: Jens T. Siveke, Rui Fang, Phyllis Fung-Yi Cheung, Jiajin Yang, Juanfei Peng, Kristina Althoff, Konstantinos Savvatakis. Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1598.

Abstract 254: Triggering head and neck cancer cell cornification leads to mucosa-like differentiation, chromatin remodeling, and loss of cell malignancy

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common tumor disease worldwide with a death rate of about 50%. As mortality and morbidity of affected patients were not substantially improved in recent decades, new treatment options are urgently needed. HNSCCs that are negative for the human papillomavirus are characterized by areas of differentiated keratinized tissue. Dissecting the mechanisms regulating this terminal differentiation of HNSCC cells may unravel targets with high potential for anti-tumor therapy. Using primary tumor-initiating spheroid cells, we established a model of HNSCC cell differentiation by cornification and observed a deprivation of cell malignancy leading to the adhesion of spheroid cells, loss of single-cell cloning capacity, and diminished tumor-initiating potential in immunodeficient mice. Analysis of our differentiation model employing ATAC-seq, RNA-seq, and proteomics showed an increased accessibility of small promoter regions despite overall genome closure, activation of a wound healing-associated signaling program, and upregulation of the stress keratin 17 (KRT17) as well as cornification markers including SPRR3. These results suggest that the differentiation of HNSCC cells resembles the differentiation process in other stem cell-maintained tissue systems. Multi-marker immunofluorescence analysis of human tissue revealed a reversion of the role of KRT17 from a basal stem-cell marker in normal mucosa to an early differentiation marker in HNSCC tissue and dysplastic mucosa preceding cornification. This proposes KRT17 as potential biomarker for HNSCC prevention screening. Human tissue analysis identified distinct cell differentiation states shared between HNSCC tissue and normal mucosa that were detected in higher and lower differentiated tumor tissue of distinct anatomical sub-locations. Cornification was observed to be induced at the interface between vital tumor tissue and necrotic, immune-infiltrated areas in human HNSCCs, suggesting a promoting influence of pro-inflammatory stimuli on cell differentiation. Treatment of tumor spheroids with inflammatory mediators resulted in cell attachment and cornification, indicating a potential strategy to stop the self-renewal of HNSCC cells. Our study thus reveals that the targeted differentiation of HNSCC tumor-initiating cells could be used as a future therapy approach against squamous cell carcinomas of the head and neck and other tissues. Citation Format: Felix Oppel, Sarah Gendreizig, Laura Martinez-Ruiz, Javier Florido, Alba López-Rodríguez, Harkiren Pabla, Lakshna Loganathan, Leonie Hose, Philipp Kühnel, Pascal Schmidt, Matthias Schürmann, Judith M. Neumann, Flavian Viyof Ful, Lars Uwe Scholtz, Ingo Todt, Ligum Dina, Frank Brasch, Niehaus Karsten, Germaine Escames, Tobias Busche, Jörn Kalinowski, Peter Goon, Holger Sudhoff. Triggering head and neck cancer cell cornification leads to mucosa-like differentiation, chromatin remodeling, and loss of cell malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 254.

MiRNA-27a-3p is involved in the plasticity of differentiated hepatocytes

BackgroundEpigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, are highly involved in the regulation of hepatocyte viability, proliferation, and plasticity. We have previously demonstrated that repression of H3K27 methylation in differentiated hepatic HepaRG cells by treatment with GSK-J4, an inhibitor of JMJD3 and UTX H3K27 demethylase activity, changed their phenotype, inducing differentiated hepatocytes to proliferate. In addition to the epigenetic enzymatic role in the regulation of the retro-differentiation process, emerging evidence indicate that microRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. Hence, the aim of this work is to investigate the impact of H3K27 methylation on miRNAs expression profile and its role in the regulation of the differentiation status of human hepatic progenitors HepaRG cells. MethodsA miRNA-sequencing was carried out in differentiated HepaRG cells treated or not with GSK-J4. Target searching and Gene Ontology analysis were performed to identify the molecular processes modulated by differentially expressed miRNAs. The biological functions of selected miRNAs was further investigated by transfection of miRNAs inhibitors or mimics in differentiated HepaRG cells followed by qPCR analysis, albumin ELISA assay, CD49a FACS analysis and EdU staining. ResultsWe identified 12 miRNAs modulated by GSK-J4; among these, miR-27a-3p and miR- 423-5p influenced the expression of several proliferation genes in differentiated HepaRG cells. MiR-27a-3p overexpression increased the number of hepatic cells reentering proliferation. Interestingly, both miR-27a-3p and miR-423-5p did not affect the expression levels of genes involved in the differentiation of progenitors HepaRG cells. ConclusionsModulation of H3K27me3 methylation in differentiated HepaRG cells, by GSK-J4 treatment, influenced miRNA’ s expression profile pushing liver cells towards a proliferating phenotype. We demonstrated the involvement of miR-27a-3p in reinducing proliferation of differentiated hepatocytes suggesting a potential role in liver plasticity.

CAR-T cell expansion platforms yield distinct T cell differentiation states

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.

Reactive oxygen species and its role in pathogenesis and resistance to therapy in acute myeloid leukemia.

Relapse following a short clinical response to therapy is the major challenge for the management of acute myeloid leukemia (AML) patients. Leukemic stem cells (LSC), as the source of relapse, have been investigated for their metabolic preferences and their alterations at the time of relapse. As LSC rely on oxidative phosphorylation (OXPHOS) for energy requirement, reactive oxygen species (ROS), as by-products of OXPHOS, have been investigated for their role in the effectiveness of the standard AML therapy. Increased levels of non-mitochondrial ROS, generated by nicotinamide adenine dinucleotide phosphate oxidase, in a subgroup of AML patients add to the complexity of studying ROS. Although there are various studies presenting the contribution of ROS to AML pathogenesis, resistance, and its inhibition or activation as a target, a model that can clearly explain its role in AML has not been conceptualized. This is due to the heterogeneity of AML, the dynamics of ROS production, which is influenced by factors such as the type of treatment, cell differentiation state, mitochondrial activity, and also the heterogeneous generation of non-mitochondrial ROS and limited available data on their interaction with the microenvironment. This review summarizes these challenges and the recent progress in this field.

Hindbrain boundaries as niches of neural progenitor and stem cells regulated by the extracellular matrix proteoglycan chondroitin sulphate.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

Molecular mechanisms of pancreatic cancer liver metastasis: the role of PAK2.

Pancreatic cancer remains an extremely malignant digestive tract tumor, posing a significant global public health burden. Patients with pancreatic cancer, once metastasis occurs, lose all hope of cure, and prognosis is extremely poor. It is important to investigate liver metastasis of Pancreatic cancer in depth, not just because it is the most common form of metastasis in pancreatic cancer, but also because it is crucial for treatment planning and prognosis assessment. This study aims to delve into the mechanisms of pancreatic cancer liver metastasis, with the goal of providing crucial scientific groundwork for the development of future treatment methods and drugs. We explored the mechanisms of pancreatic cancer liver metastasis using single-cell sequencing data (GSE155698 and GSE154778) and bulk data (GSE71729, GSE19279, TCGA-PAAD). Initially, Seurat package was employed for single-cell data processing to obtain expression matrices for primary pancreatic cancer lesions and liver metastatic lesions. Subsequently, high-dimensional weighted gene co-expression network analysis (hdWGCNA) was used to identify genes associated with liver metastasis. Machine learning algorithms and COX regression models were employed to further screen genes related to patient prognosis. Informed by both biological understanding and the outcomes of algorithms, we meticulously identified the ultimate set of liver metastasis-related gene (LRG). In the study of LRG genes, various databases were utilized to validate their association with pancreatic cancer liver metastasis. In order to analyze the effects of these agents on tumor microenvironment, we conducted an in-depth analysis, including changes in signaling pathways (GSVA), cell differentiation (pseudo-temporal analysis), cell communication networks (cell communication analysis), and downstream transcription factors (transcription factor activity prediction). Additionally, drug sensitivity analysis and metabolic analysis were performed to reveal the effects of LRG on gemcitabine resistance and metabolic pathways. Finally, functional experiments were conducted by silencing the expression of LRG in PANC-1 and Bx-PC-3 cells to validate its influence to proliferation and invasiveness on PANC-1 and Bx-PC-3 cells. Through a series of algorithmic filters, we identified PAK2 as a key gene promoting pancreatic cancer liver metastasis. GSVA analysis elucidated the activation of the TGF-beta signaling pathway by PAK2 to promote the occurrence of liver metastasis. Pseudo-temporal analysis revealed a significant correlation between PAK2 expression and the lower differentiation status of pancreatic cancer cells. Cell communication analysis revealed that overexpression of PAK2 promotes communication between cancer cells and the tumor microenvironment. Transcription factor activity prediction displayed the transcription factor network regulated by PAK2. Drug sensitivity analysis and metabolic analysis revealed the impact of PAK2 on gemcitabine resistance and metabolic pathways. CCK8 experiments showed that silencing PAK2 led to a decrease in the proliferative capacity of pancreatic cancer cells and scratch experiments demonstrated that low expression of PAK2 decreased invasion capability in pancreatic cancer cells. Flow cytometry reveals that PAK2 significantly inhibited apoptosis in pancreatic cancer cell lines. Molecules related to the TGF-beta pathway decreased with the inhibition of PAK2, and there were corresponding significant changes in molecules associated with EMT. PAK2 facilitated the angiogenic potential of cancer cells and promotes the epithelial-mesenchymal transition process by activating the TGF-beta signaling pathway. Simultaneously, it decreased the differentiation level of cancer cells, consequently enhancing their malignancy. Additionally, PAK2 fostered communication between cancer cells and the tumor microenvironment, augments cancer cell chemoresistance, and modulates energy metabolism pathways. In summary, PAK2 emerged as a pivotal gene orchestrating pancreatic cancer liver metastasis. Intervening in the expression of PAK2 may offer a promising therapeutic strategy for preventing liver metastasis of pancreatic cancer and improving its prognosis.

Glycan Modifications as Regulators of Stem Cell Fate.

Glycosylation is a process where proteins or lipids are modified with glycans. The presence of glycans determines the structure, stability, and localization of glycoproteins, thereby impacting various biological processes, including embryogenesis, intercellular communication, and disease progression. Glycans can influence stem cell behavior by modulating signaling molecules that govern the critical aspects of self-renewal and differentiation. Furthermore, being located at the cell surface, glycans are utilized as markers for stem cell pluripotency and differentiation state determination. This review aims to provide a comprehensive overview of the current literature, focusing on the effect of glycans on stem cells with a reflection on the application of synthetic glycans in directing stem cell differentiation. Additionally, this review will serve as a primer for researchers seeking a deeper understanding of how synthetic glycans can be used to control stem cell differentiation, which may help establish new approaches to guide stem cell differentiation into specific lineages. Ultimately, this knowledge can facilitate the identification of efficient strategies for advancing stem cell-based therapeutic interventions.

A new perspective on intervertebral disc calcification-from bench to bedside.

Disc degeneration primarily contributes to chronic low back and neck pain. Consequently, there is an urgent need to understand the spectrum of disc degeneration phenotypes such as fibrosis, ectopic calcification, herniation, or mixed phenotypes. Amongst these phenotypes, disc calcification is the least studied. Ectopic calcification, by definition, is the pathological mineralization of soft tissues, widely studied in the context of conditions that afflict vasculature, skin, and cartilage. Clinically, disc calcification is associated with poor surgical outcomes and back pain refractory to conservative treatment. It is frequently seen as a consequence of disc aging and progressive degeneration but exhibits unique molecular and morphological characteristics: hypertrophic chondrocyte-like cell differentiation; TNAP, ENPP1, and ANK upregulation; cell death; altered Pi and PPi homeostasis; and local inflammation. Recent studies in mouse models have provided a better understanding of the mechanisms underlying this phenotype. It is essential to recognize that the presentation and nature of mineralization differ between AF, NP, and EP compartments. Moreover, the combination of anatomic location, genetics, and environmental stressors, such as aging or trauma, govern the predisposition to calcification. Lastly, the systemic regulation of calcium and Pi metabolism is less important than the local activity of PPi modulated by the ANK-ENPP1 axis, along with disc cell death and differentiation status. While there is limited understanding of this phenotype, understanding the molecular pathways governing local intervertebral disc calcification may lead to developing disease-modifying drugs and better clinical management of degeneration-related pathologies.

Text mining for contexts and relationships in cancer genomics literature.

Scientific advances build on the findings of existing research. The 2001 publication of the human genome has led to the production of huge volumes of literature exploring the context-specific functions and interactions of genes. Technology is needed to perform large-scale text mining of research papers to extract the reported actions of genes in specific experimental contexts and cell states, such as cancer, thereby facilitating the design of new therapeutic strategies. We present a new corpus and Text Mining methodology that can accurately identify and extract the most important details of cancer genomics experiments from biomedical texts. We build a Named Entity Recognition model that accurately extracts relevant experiment details from PubMed abstract text, and a second model that identifies the relationships between them. This system outperforms earlier models and enables the analysis of gene function in diverse and dynamically evolving experimental contexts. Code and data are available here: https://github.com/cambridgeltl/functional-genomics-ie.

Machine learning classification of cellular states based on the impedance features derived from microfluidic single-cell impedance flow cytometry.

Mitosis is a crucial biological process where a parental cell undergoes precisely controlled functional phases and divides into two daughter cells. Some drugs can inhibit cell mitosis, for instance, the anti-cancer drugs interacting with the tumor cell proliferation and leading to mitosis arrest at a specific phase or cell death eventually. Combining machine learning with microfluidic impedance flow cytometry (IFC) offers a concise way for label-free and high-throughput classification of drug-treated cells at single-cell level. IFC-based single-cell analysis generates a large amount of data related to the cell electrophysiology parameters, and machine learning helps establish correlations between these data and specific cell states. This work demonstrates the application of machine learning for cell state classification, including the binary differentiations between the G1/S and apoptosis states and between the G2/M and apoptosis states, as well as the classification of three subpopulations comprising a subgroup insensitive to the drug beyond the two drug-induced states of G2/M arrest and apoptosis. The impedance amplitudes and phases used as input features for the model training were extracted from the IFC-measured datasets for the drug-treated tumor cells. The deep neural network (DNN) model was exploited here with the structure (e.g., hidden layer number and neuron number in each layer) optimized for each given cell type and drug. For the H1650 cells, we obtained an accuracy of 78.51% for classification between the G1/S and apoptosis states and 82.55% for the G2/M and apoptosis states. For HeLa cells, we achieved a high accuracy of 96.94% for classification between the G2/M and apoptosis states, both of which were induced by taxol treatment. Even higher accuracy approaching 100% was achieved for the vinblastine-treated HeLa cells for the differentiation between the viable and non-viable states, and between the G2/M and apoptosis states. We also demonstrate the capability of the DNN model for high-accuracy classification of the three subpopulations in a complete cell sample treated by taxol or vinblastine.

A comprehensive exploration of the heterogeneity of immune cells in Han and Zang systemic lupus erythematosus patients via single-cell RNA sequencing

Systemic Lupus Erythematosus (SLE) is an autoimmune sickness with unclear pathogenesis. The goal of this research was to reveal the heterogeneity of immune cells in SLE patients of Han and Zang nationality by single-cell RNA sequencing (scRNA-seq) and bioinformatics profiling. MethodsA total of 94,102 peripheral blood mononuclear cells (PBMCs) from six volunteers with SLE (3 Zang, 3 Han) and six healthy controls were first conducted through scRNA-seq analysis. The immune cell subsets in the pathogenesis of SLE were analyzed as well. Real-time quantitative PCR (RT-qPCR) was applied to confirm the results of sc-RNA seq analysis. ResultsFor the Tibetan samples, the ratios of Naïve CD4 RPS4Y1 cells, Naïve CD4 cells, Memory BC CD24 and Memory BC differed significantly between the SLE and control samples, while that of CD8 CTL MAL cells was significantly different between the two groups in Han nationality samples. Variable differentiation states of CD8 CTL MAL cells, CD8 CTL GZMK cells, and Naïve CD4 cells were detected through pseudotime analysis. Moreover, T-cell receptor (TCR) abundance was notably higher in Tibetan SLE specimens than that in controls, while B-cell receptor (BCR) abundance in Tibetan and Han samples was higher than in control groups. ConclusionsIn summary, the immune cellular heterogeneity of SLE patients both Han and Zang nationality was explored based on various bioinformatics approaches, providing new perspectives for immunological characteristics of SLE among different ethnic groups.