Bulk Population Research Articles

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Complete suspension culture of human induced pluripotent stem cells supplemented with suppressors of spontaneous differentiation.

Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.

Efficient CRISPR/Cas9 Knock-in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells.

Precise understanding of temporally controlled protein-protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR-based knock-in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock-in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9-mediated knock-in. We address knock-in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co-expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single-cell or bulk populations with the desired knock-in, particularly when knock-in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N- or C-termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock-in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Choosing an optimal knock-in target site and single guide RNA (sgRNA) design Basic Protocol 2: Assessment of Cas9 editing efficiency at the desired B cell genomic knock-in site Basic Protocol 3: Cloning the sgRNA dual guide construct Basic Protocol 4: Repair template design and cloning Basic Protocol 5: Electroporation and selection of engineered B cells Basic Protocol 6: Single-cell cloning of engineered B cells.

Single-cell eQTL mapping in yeast reveals a tradeoff between growth and reproduction.

Expression quantitative trait loci (eQTLs) provide a key bridge between noncoding DNA sequence variants and organismal traits. The effects of eQTLs can differ among tissues, cell types, and cellular states, but these differences are obscured by gene expression measurements in bulk populations. We developed a one-pot approach to map eQTLs in Saccharomyces cerevisiae by single-cell RNA sequencing (scRNA-seq) and applied it to over 100,000 single cells from three crosses. We used scRNA-seq data to genotype each cell, measure gene expression, and classify the cells by cell-cycle stage. We mapped thousands of local and distant eQTLs and identified interactions between eQTL effects and cell-cycle stages. We took advantage of single-cell expression information to identify hundreds of genes with allele-specific effects on expression noise. We used cell-cycle stage classification to map 20 loci that influence cell-cycle progression. One of these loci influenced the expression of genes involved in the mating response. We showed that the effects of this locus arise from a common variant (W82R) in the gene GPA1 , which encodes a signaling protein that negatively regulates the mating pathway. The 82R allele increases mating efficiency at the cost of slower cell-cycle progression and is associated with a higher rate of outcrossing in nature. Our results provide a more granular picture of the effects of genetic variants on gene expression and downstream traits.

Lower hybrid and solitary waves in dusk flank region of the Earth’s magnetosphere

A variety of plasma waves have been detected in the vicinity of the magnetopause by various spacecraft missions. In this paper, utilizing high-resolution data from the Magnetospheric Multiscale (MMS) mission, we present new observations of simultaneous lower hybrid and ion solitary waves in the dusk flank region of Earth’s magnetosphere. All four MMS spacecraft consistently observed this wave activity during their traversal from Earth’s magnetosphere to the magnetosheath. Our analysis suggests that the lower hybrid drift waves, driven by lower-hybrid drift instability, were observed in correlation with density gradients. Furthermore, the analysis indicates that the entire ion bulk population drifts, which drives ion solitary waves. It is found that these waves play a crucial role in particle heating in the dusk flank region of Earth’s magnetosphere.

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription.

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

Quantifying the Diversity of an Atmospheric Aerosol Population in an Arctic Oil Field on a Single‐Particle Level

AbstractAs the Arctic rapidly warms, sea ice extent is decreasing and oil and gas extraction activities are expanding. Local combustion emissions affect the Arctic atmospheric aerosol chemical mixing state (the distribution of chemical species across the aerosol population), which impacts climate‐relevant properties. Bulk and single‐particle measurements of submicron aerosols were conducted at Oliktok Point, Alaska within the North Slope of Alaska oil fields. In this work, we quantify aerosol diversity using online single‐particle mass spectrometry data (32,880 individual particles), offline single‐particle microscopy data (20,912 individual particles), and online bulk aerosol mass spectrometry and aethalometer data. This method was used to derive individual particle mass fractions for both refractory and non‐refractory material within distinct particle types. Single‐particle, average single‐particle, and bulk population diversities (Di, Dα, Dγ, respectively) and mixing state indices (χ) were calculated for the data set. Calculated Di values were generally low (2.2 ± 0.6), as individual particle masses were dominated by a few chemical species of interest. Aged aerosol particles (those internally mixed with nitrate and/or sulfate) exhibited higher Di values (>3) compared to recently emitted (fresh) aerosol particles. During oil field plume periods, Dα values approached three due to the abundance of diesel combustion particles, which were rich in sulfate, black carbon, and organic aerosol. Overall, the submicron aerosol population within the Arctic oil fields was found to be relatively externally mixed (χ < 50%), due to the constant local emissions within oil fields combining with background aerosol and locally emitted sea spray aerosol at the coastal site.

Splicing variants of versican in CD133+/CD44+ prostate cancer stem cells

A cancer mass is composed of a heterogeneous group of cells, a small part of which constitutes the cancer stem cells since they are less differentiated and have a high capacity to develop cancer. Versican is an extracellular matrix protein located in many human tissues. The mRNA of versican has been shown to have “splicing patterns” as detected by RT-PCR, northern blot analysis, and cDNA sequencing. Based on this knowledge this study aims to reveal the splice variants of versican molecules, which are thought to be involved in the pathogenesis of the DU-145 human prostatic carcinoma cell line and prostatic cancer stem cells isolated from this cell line. In this study, RWPE-1 normal prostatic and DU-145 human prostate cancer cell lines have been used. Prostatic cancer stem cells and the remaining group of non-prostatic-cancer stem cells (bulk population) were isolated according to their CD133+/CD44+. RNA was isolated in all groups, and sequence analysis was accomplished for splicing variants by Illumina NextSeq 500 sequencing system. The results were analyzed by bioinformatic evaluation. As five isoforms of the versican gene in the differential transcript expression are analyzed, it was observed that a significant change was only found in the isoforms Versican 0 and Versican 1. In this study, we explored the function of this molecule which we think to be effective in cancer progression, and suggested that more valuable results can be obtained after the accomplishment of in vivo experiments.

The Origin of Young Stellar Populations in NGC 1783: Accretion of External Stars

The presence of young stellar populations in the Large Magellanic Cloud cluster NGC 1783 has caught significant attention, with suggestions ranging from it being a genuine secondary stellar generation to a population of blue straggler stars or simply contamination from background stars. Thanks to multiepoch observations with the Hubble Space Telescope, proper motions for stars within the field of NGC 1783 have been derived, thus allowing accurate cluster membership determination. Here, we report that the younger stars within NGC 1783 indeed belong to the cluster and their spatial distribution is more extended compared to the bulk of the older stellar population, consistent with previous studies. Through N-body simulations, we demonstrate that the observed characteristics of the younger stars cannot be explained solely by blue straggler stars in the context of the isolated dynamical evolution of NGC 1783. Instead, accretion of the external, low-mass stellar system can better account for both the inverse spatial concentration and the radial velocity isotropy of the younger stars. We propose that NGC 1783 may have accreted external stars from low-mass stellar systems, resulting in a mixture of external younger stars and blue straggler stars from the older bulk population, thereby accounting for the characteristics of the younger sequence.

Reliability analysis of deep space satellites launched 1991–2020: Bulk population and deployable satellite performance analysis

AbstractFlight data for deep space satellites launched and operated between 1991 and 2020 is analyzed to generate various reliability metrics. Satellite reliability is first estimated by the Kaplan‐Meier estimator, then parameterized through the Weibull distribution. This general process is applied to a general satellite data set that included all deep space satellites launched between 1991 and 2020, as well as two data subsets. One subset focuses on deployable satellites, while the other introduces a methodology of normalizing satellite lifetimes by satellite design life. Results from the general data set prove deep space satellites suffer from infant mortality while the results from the deployable data subset show deployable deep space satellites are only reliable over short periods of time. Results from the design life normalized data set give promising results, with satellites having a relatively high chance of reaching their design life. Available information regarding specific modes of failure is also leveraged to generate a percent contribution to overall satellite failure for eight distinct failure modes. Satellite failure due to crashing, in‐space propulsion failure, and telemetry system failure are proven to drive both early in life failure and later in life failure, making them the main causes of decreased reliability.

Ganglioside SSEA-4 in Ewing sarcoma marks a tumor cell population with aggressive features and is a potential cell-surface immune target

Carbohydrate markers of immature cells during prenatal human development can be aberrantly expressed in cancers and deserve evaluation as immune targets. A candidate target in Ewing sarcoma is the globo-series ganglioside stage-specific embryonic antigen-4 (SSEA-4). We detected SSEA-4 expression on the cell surface of all of 14 EwS cell lines and in 21 of 31 (68%) primary EwS tumor biopsies. Among paired subpopulations of tumor cells with low versus high SSEA-4 expression, SSEA-4high expression was significantly and consistently associated with functional characteristics of tumor aggressiveness, including higher cell proliferation, colony formation, chemoresistance and propensity to migrate. SSEA-4low versus SSEA-4high expression was not related to expression levels of the EWSR1-FLI1 fusion transcript or markers of epithelial/mesenchymal plasticity. SSEA-4low cells selected from bulk populations regained higher SSEA-4 expression in vitro and during in vivo tumor growth in a murine xenograft model. T cells engineered to express SSEA-4-specific chimeric antigen receptors (CARs) specifically interacted with SSEA-4 positive EwS cells and exerted effective antigen-specific tumor cell lysis in vitro. In conclusion, with its stable expression and functional significance in EwS, SSEA-4 is an attractive therapeutic immune target in this cancer that deserves further evaluation for clinical translation.

Abstract 25: Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features

Abstract Introduction: Autologous TIL cell therapies such as lifileucel have demonstrated durable responses in patients (pts) with various solid tumors including melanoma and lung cancer. The TIL drug product (DP) phenotype was previously assessed and extensively characterized based on the bulk population. The current research further characterizes the TIL DP at a single-cell level using high-dimension multimodal sequencing and explores features that have a potential to inform future development of TIL therapies. Methods: Manufactured lifileucel TIL DP from 20 pts (best overall response [BOR]: 6 complete response [CR], 4 partial response, 5 stable disease, 5 progressive disease [PD]) from the registrational melanoma C144-1 trial were analyzed using single-cell RNA and T-cell receptor (TCR) sequencing, resulting in 153K single-cell transcriptomes. To aid with cell-type annotation, 130 surface proteins on TIL DP samples from a subset of 4 pts (BOR: 2 CR, 2 PD) were detected via Cellular Indexing of Transcriptomes and Epitopes by sequencing. Multimodal weighted nearest-neighbor analysis combined with manual cluster annotation (via known marker genes and proteins), cell-type-specific gene signatures, and automated annotation were used to develop a single-cell TIL (scTIL) reference map, resulting in high-resolution subsets of cells. TCR clonotypes from lifileucel TIL DP were compared to those from respective pretreatment tumors previously identified by bulk TCR sequencing. Results: Mapping of the full dataset to the novel scTIL reference revealed a CD8+ TIL-dominant composition of responder TIL DP. TIL from pts with BOR PD exhibited reduced CD8+ effector memory (Tem)-like and resident memory (Trm)-like T cells. Differential expression analysis showed that responder CD8+ TIL had increased expression of TCR signaling genes while responder CD4+ TIL exhibited gene expression consistent with a more cytotoxic-like phenotype. Relative to responders, CD8+ TIL of pts with BOR PD had lower expression of the CD39−CD69− (stem-like) gene signature and higher expression of the CD39+CD69+ (terminal differentiation) gene signature. Pseudotime trajectory analysis found that both CD8+ and CD4+ TIL of pts with BOR PD were more terminally differentiated. Single-cell TCR sequencing revealed that CD8+ T-cell clusters had greater clonal overlap than CD4+ T-cell clusters. Expanded clonotypes in pretreatment tumor tissues were more prevalent in TIL TCR repertoires of responders and were more likely to be expressed by CD8+ (specifically CD8+ Tem- and Trm-like TIL) than CD4+ T cells. Conclusions: Collectively, these results shed additional light on the cellular and molecular characteristics of ex vivo expanded lifileucel TIL DP and signal additional features with a potential to guide future TIL product development. Citation Format: Joe Dean, Joe Yglesias, Hequn Yin, Rongsu Qi. Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features [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 25.

Abstract 6802: CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells

Abstract Chimeric antigen receptor (CAR)-engineered induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have shown a favorable safety profile and early evidence of clinical efficacy in the treatment of patients with certain hematologic malignancies. CAR-iNK cells retain phenotypic and functional similarities to primary natural killer cells, with additional capabilities for recognition of tumors. For the treatment of cancers that are located in the bone marrow, it is critical that iNK cells are able to migrate efficiently to relevant sites of disease. CXCR4 (C-X-C chemokine receptor type 4) is a chemokine receptor specific for stromal-derived-factor-1 (SDF-1), and mediates the migration of hematopoietic cells to the bone marrow upon binding SDF-1. Here we describe novel engineering of iPSCs resulting in efficient migration and efficacy of iNK cells. iPSC were sequentially engineered at defined loci with Crispr/MAD7/HDR to incorporate Allo-EvasionTM edits, a CAR targeting antigens expressed on different hematological malignancies, constitutive interleukin-15 (IL-15) cytokine support, with and without wild-type CXCR4. iPSCs were then differentiated into natural killer cells using a proprietary differentiation process. Cytolysis of a panel of target cells by iNK was evaluated using an Incucyte-based killing assay. Anti-tumor efficacy was tested in disseminated xenograft models of hematological malignancies following intravenous injection of engineered iNK cells. Additionally, in vivo migration was evaluated using immunohistochemistry (IHC) and flow cytometry (FACS) of blood, bone marrow, and other organs. Engineering with CXCR4 resulted in a bulk population of iNK with a heterogenous expression of CXCR4. No significant impact of CXCR4 engineering was detected on iPSC differentiation, phenotype, or iNK yield. iNKCXCR4+ and iNKCXCR4− cells had equivalent target tumor cell cytolysis in vitro, indicating the CXCR4 transgene does not affect killing potential. In vivo, iNKCXCR4+ cells migrated to the bone marrow of both tumor-bearing and non-tumor-bearing animals, and the presence of these cells was enhanced in tumor-bearing mice, indicating CXCR4-mediated migration and possibly CAR-mediated expansion and/or retention of iNK. Migration to the bone marrow was not observed with iNKCXCR4− cells. As a result, iNKCXCR4+ cells significantly impacted anti-tumor efficacy, effectively eliminating bone disease. We have demonstrated the CAR-specific killing and enhanced iNK migration and subsequent tumor killing in the bone marrow, with the addition of a CXCR4 transgene in iPSC-derived NK cells. The addition of the CXCR4 transgene in iPSC-derived effector cells may allow for a higher therapeutic response in axial-skeletal located tumors. Citation Format: Hillary J. Millar, David Walker, Liam R. Campion, Ciara Budd, Aarti Kolluri, Harsh Sharma, Buddha Gurung, Jill M. Carton, Daniel J. Perry, Nicholas Alexander, Hyam Levitsky, Michael Naso, Barry A. Morse. CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer 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 6802.

Abstract 4300: Large-scale, low-cost, and accurate measurement of cancer evolutionary dynamics from clinical patient samples

Abstract Introduction: Cancer is a dynamic evolutionary process; thus, the evolutionary history of a cancer should strongly influence its future trajectory and so the clinical outcome of patients. However, accurately assessing an individual cancer’s evolutionary history in a scalable and clinically relevant manner is currently an open problem. To address this, we introduce a novel methodology (EVOFLUx) to quantitively infer the history of a cancer from single time-point bulk samples using inexpensive, standard methylation arrays. Description of Methodology: We recently identified fluctuating CpGs (fCpGs), which are CpGs that neutrally and stochastically change methylation state over time in individual cells, uniquely barcoding cells and thus enabling high temporal-resolution lineage tracing approaches. In a bulk population of cancer cells, the distribution of fCpG methylation is determined by the evolution of the population and thus encode a cancer’s past clonal dynamics. Here, we have developed a general computational approach to model how the patterns of fCpGs vary according to the evolutionary history of a growing cancer, and a Bayesian inference method to infer these parameters from data. Results We applied EVOFLUx to quantify the evolutionary dynamics of 1,976 lymphoid cancers, finding that the tumor growth rates, malignancy age and epimutation rates varied by orders of magnitude across disease types. In addition, EVOFLUx allowed us to test for the presence of ongoing subclonal selection and validated our inference with matched whole exome sequencing data. Alongside characterizing the evolutionary dynamics within single bulk samples, fCpGs also allow for the inference of the phylogenetic relationships between longitudinal samples, which we validated with whole genome sequencing. Within cancer types, more aggressive subtypes typically had elevated growth rates. In our clinically annotated cohort of chronic lymphocytic leukemia (CLL) samples, we found that patient-specific evolutionary dynamics are strongly associated with outcome. Conclusions: Hence, we present a powerful new method that uses widely available, low-cost bulk DNA methylation data to precisely measures cancer evolutionary dynamics in patient samples with clinical implications. Citation Format: Calum Gabbutt, Martí Duran-Ferrer, Heather Grant, Diego Mallo, Ferran Nadeu, Jacob Househam, Neus Villamor, Olga Krali, Jessica Nordlund, Thorsten Zenz, Elias Campo, Armando Lopez-Guillermo, Jude Fitzgibbon, Chris P. Barnes, Darryl Shibata, José I. Martin-Subero, Trevor A. Graham. Large-scale, low-cost, and accurate measurement of cancer evolutionary dynamics from clinical patient samples [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 4300.

Abstract B068: FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models

Abstract The unfavorable prognosis of high-grade serous ovarian cancer, HGSOC, may be driven by a small subset of ovarian cancer stem cells (OCSC), which are endowed with tumor-initiating ability and are intrinsically resistant to cytotoxic treatments. These characteristics are sustained by signals coming from the tumor microenvironment (TME). As a common site of HGSOC presentation is the omentum, we partially recapitulated the architecture of human omentum in vitro using 3D co-cultures of primary human omental fibroblasts and mesothelial cells. Patient-derived HGSOC cells were then integrated in these organotypic models, either as bulk cancer cells or as OCSC from the same patients. After co-culture in this model, malignant cells were re-isolated by FACS sorting and subjected to whole-transcriptome RNA sequencing, comparing them to control cells that have not been in contact with the in vitro TME. Our data revealed transcriptional reprogramming induced by the in vitro TME in HGSOC cells. We found a number of pathways differentially regulated in the bulk population of OC cells vs OCSC, shedding light on the impact of the TME on OC stemness. Moreover, we identified a panel of transcription factors (TFs) specifically altered by the in vitro TME in OCSC. These TF-regulated gene networks represent OCSC vulnerabilities and, hence, potential targets in relapsing tumors. Among these, we focused on FOXM1, a TF with pleiotropic functions in cancer cells, whose activity is altered in 84% of ovarian cancer patients. The FOXM1 pathway was strongly induced by the in vitro TME specifically in OCSC. Mechanistically, the contact with the in vitro TME triggered a FAK/YAP axis which, in turn, accounted for the induction of FOXM1 and its downstream target genes. Moreover, the pharmacological inhibition of FOXM1 significantly impaired OCSC fitness, supporting, on one hand, the key role of TME in orchestrating OC stemness and suggesting, on the other, a new approach for the eradication of OCSC from the omental niche. Overall, our patient-derived organotypic platform offers the opportunity to unravel the impact of the TME on OCSC at the molecular level in experimental models with clinical relevance and can be exploited to design innovative HGSOC-eradicating treatments. Citation Format: Chiara Battistini, Hilary A. Kenny, Valentina Nieddu, Valentina Melocchi, Alessandra Decio, Beatrice Malacrida, Alessia Gatto, Fabrizio Bianchi, Raffaella Giavazzi, Nicoletta Colombo, Frances R. Balkwill, Ernst Lengyel, Ugo Cavallaro. FOXM1 mediates the crosstalk between tumor microenvironment and ovarian cancer stem cells as revealed by patient-derived organotypic models [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B068.

Inferring bacterial interspecific interactions from microcolony growth expansion.

Bacterial species interactions significantly shape growth and behavior in communities, determining the emergence of community functions. Typically, these interactions are studied through bulk population measurements, overlooking the role of cell-to-cell variability and spatial context. This study uses real-time surface growth measurements of thousands of sparsely positioned microcolonies to investigate interactions and kinetic variations in monocultures and cocultures of Pseudomonas putida and P. veronii under substrate competition (succinate) or substrate independence (d-mannitol and putrescine). In monoculture, microcolonies exhibited expected substrate-dependent expansion rates, but individual colony sizes were affected by founder cell density, spatial positioning, growth rates, and lag times. In coculture, substrate competition favored P. putida, but unexpectedly, reduced the maximum growth rates of both species. In contrast, 10% of P. veronii microcolonies under competition grew larger than expected, likely due to founder cell phenotypic variation and stochastic spatial positioning. These effects were alleviated under substrate independence. A linear relationship between founder cell ratios and final colony area ratios in local neighborhoods (6.5-65µm radius) was observed in coculture, with its slope reflecting interaction type and strength. Measured slopes in the P. putida to P. veronii biomass ratio under competition were one-third reduced compared to kinetic predictions using a cell-agent growth model, which exometabolite analysis and simulations suggested may be due to metabolite cross-feeding or inhibitory compound production. This indicates additional factors beyond inherent monoculture growth kinetics driving spatial interactions. Overall, the study demonstrates how microcolony growth experiments offer valuable insights into bacterial interactions, from local to community-level dynamics.

DNA Sequencing of CD138 Cell Population Reveals TP53 and RAS-MAPK Mutations in Multiple Myeloma at Diagnosis.

Multiple myeloma is a hematologic neoplasm caused by abnormal proliferation of plasma cells. Sequencing studies suggest that plasma cell disorders are caused by both cytogenetic abnormalities and oncogene mutations. Therefore, it is necessary to detect molecular abnormalities to improve the diagnosis and management of MM. The main purpose of this study is to determine whether NGS, in addition to cytogenetics, can influence risk stratification and management. Additionally, we aim to establish whether mutational analysis of the CD138 cell population is a suitable option for the characterization of MM compared to the bulk population. Following the separation of the plasma cells harvested from 35 patients newly diagnosed with MM, we performed a FISH analysis to detect the most common chromosomal abnormalities. Consecutively, we used NGS to evaluate NRAS, KRAS, BRAF, and TP53 mutations in plasma cell populations and in bone marrow samples. NGS data showed that sequencing CD138 cells provides a more sensitive approach. We identified several variants in BRAF, KRAS, and TP53 that were not previously associated with MM. Considering that the presence of somatic mutations could influence risk stratification and therapeutic approaches of patients with MM, sensitive detection of these mutations at diagnosis is essential for optimal management of MM.

Cell metabolism: Functional and phenotypic single cell approaches.

Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.

Performances of F3 and F4 Bulk Populations in Cotton (Gossypium hirsutum L.)

The experiment was laid out in 2020 to compare the performance of thirty-seven F3 and F4 multi-parental bulk populations of cotton, including comparative cultivars, and to assess the heritability of traits studied. The differences within generations were significant for seed cotton yield, ginning out-turn, fiber quality, and nep fragments. The mean fiber strength of the F4 generation showed significant performance compared with F3. The broad sense heritability was high for ginning out-turn, fiber length, and fiber strength. The normal distribution for F3 and F4 generations due to non-significant skewness and kurtosis values indicated that there were no epistatic effects on the heritability of traits studied. Eight F4 lines were selected for transfer to F5 generation according to optimization in terms of desired traits.

Efficient gene editing in induced pluripotent stem cells enabled by an inducible adenine base editor with tunable expression

The preferred method for disease modeling using induced pluripotent stem cells (iPSCs) is to generate isogenic cell lines by correcting or introducing pathogenic mutations. Base editing enables the precise installation of point mutations at specific genomic locations without the need for deleterious double-strand breaks used in the CRISPR-Cas9 gene editing methods. We created a bulk population of iPSCs that homogeneously express ABE8e adenine base editor enzyme under a doxycycline-inducible expression system at the AAVS1 safe harbor locus. These cells enabled fast, efficient and inducible gene editing at targeted genomic regions, eliminating the need for single-cell cloning and screening to identify those with homozygous mutations. We could achieve multiplex genomic editing by creating homozygous mutations in very high efficiencies at four independent genomic loci simultaneously in AAVS1-iABE8e iPSCs, which is highly challenging with previously described methods. The inducible ABE8e expression system allows editing of the genes of interest within a specific time window, enabling temporal control of gene editing to study the cell or lineage-specific functions of genes and their molecular pathways. In summary, the inducible ABE8e system provides a fast, efficient and versatile gene-editing tool for disease modeling and functional genomic studies.